CN102665758A - Dock-and-lock (DNL) Complexes For Delivery of Interference RNA - Google Patents

Dock-and-lock (DNL) Complexes For Delivery of Interference RNA Download PDF

Info

Publication number
CN102665758A
CN102665758A CN2010800526905A CN201080052690A CN102665758A CN 102665758 A CN102665758 A CN 102665758A CN 2010800526905 A CN2010800526905 A CN 2010800526905A CN 201080052690 A CN201080052690 A CN 201080052690A CN 102665758 A CN102665758 A CN 102665758A
Authority
CN
China
Prior art keywords
seq
sirna
antibody
cancer
targeted delivery
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN2010800526905A
Other languages
Chinese (zh)
Inventor
C-H·张
D·M·戈登堡
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
IBC Pharmaceuticals Inc
Original Assignee
IBC Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US12/644,146 external-priority patent/US7981398B2/en
Priority claimed from US12/731,781 external-priority patent/US8003111B2/en
Priority claimed from US12/752,649 external-priority patent/US8034352B2/en
Priority claimed from US12/754,740 external-priority patent/US8562988B2/en
Priority claimed from US12/869,823 external-priority patent/US20110020273A1/en
Priority claimed from US12/871,345 external-priority patent/US8551480B2/en
Priority claimed from US12/915,515 external-priority patent/US20110064754A1/en
Application filed by IBC Pharmaceuticals Inc filed Critical IBC Pharmaceuticals Inc
Publication of CN102665758A publication Critical patent/CN102665758A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Public Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Described herein are compositions and methods of use of targeted delivery complexes for delivery of siRNA to a disease-associated cell, tissue or pathogen. The targeted delivery complex comprises a targeting molecule, such as an antibody or fragment thereof, conjugated to one or more siRNA carriers. In preferred embodiments the siRNA carrier is a dendrimer or protamine and the targeting molecule is an anti-cancer antibody, such as hRS7. More preferably, the antibody or fragment is rapidly internalized into the target cell to facilitate uptake of the siRNA. Most preferably, the targeted delivery complex is made by the DNL technique. The compositions and methods are of use to treat a variety of disease states, such as cancer, autoimmune disease, immune dysfunction, cardiac disease, neurologic disease, inflammatory disease or infectious disease.

Description

Be used to send butt joint locking (DNL) complex of RNA interfering
Related application
The application requires the U.S. Patent application No.12/949 of 11/18/2010 submission, 536; The U.S. Patent application No.12/915 of 10/29/2010 submission, 515; The U.S. Patent application No.12/871 of 8/30/2010 submission, 345; The U.S. Patent application No.12/869 of 8/27/2010 submission, 823; The U.S. Patent application No.12/754 of 4/6/2010 submission, 740; The U.S. Patent application No.12/752 of 4/1/2010 submission, 649; The U.S. Patent application No.12/731 of 3/25/2010 submission, 781; The U.S. Patent application No.12/644 of 12/22/2009 submission, 146 priority.
The application also requires the U.S. Provisional Patent Application No.61/267 of December in 2009 submission on the 9th; 877, the U.S. Provisional Patent Application No.61/302 that submitted on February 8th, 2010; The U.S. Provisional Patent Application No.61/414 that on November 17th, 682 and 2010 submitted to, 592 rights and interests.The text of each priority application is all incorporated this paper by reference into.
Background of invention
Invention field
The present invention relates to be used for the compositions and the method for targeted delivery RNAi material (like siRNA).Preferably, the targeted delivery complex comprises and the link coupled antibody of the carrier molecule of siRNA, antibody fragment or other targeted molecular, like protamine or dendritic.More preferably, the targeted delivery complex is through butt joint locking (dock-and-lock; DNL) technology preparation.The targeted delivery complex can comprise one or more siRNA materials for delivery to target cell or tissue.Most preferably; Send siRNA and can effectively treat disease, syndrome or disease, like cancer, autoimmune disease, immune dysfunction (for example graft versus host disease or organ transplant rejection), inflammation, infectious disease, cardiovascular disease, endocrine or metabolic disease or neurodegenerative disease.For physics, antibody or other targeted molecular combine to be produced or otherwise relevant with disease patient's condition target antigen by diseased cells or tissue.The DNL complex can comprise the carrier molecule of a plurality of copies effectively therapeutic siRNA is delivered to target cell.Antibody or other targeted molecular can be the material of quick internalization to absorb in the cell that promotes siRNA.
Association area
It is the regulatory mechanism (Fire etc., 1998, Nature 391:806-11) of gene expression in the naturally occurring control cell that RNA disturbs (RNAi).RNAi is by the inductive reticent complex of RNA (RNA-induced silencing complex; RISC) mediation and by with the interactional short dsrna molecule of catalytic RISC component argonaute starting (Rand etc., 2005, Cell 123:621-29).The type of RNAi molecule comprises microRNA (miRNA) and siRNA (siRNA).The RNAi material can combine through the complementary base pairing with messenger RNA (mRNA) and come inhibition of gene expression through PTGS.After complementary mRNA material combined, RNAi was through the cracking of the argonaute component induction mRNA molecule of RISC.MiRNA and siRNA are different aspect the degrees of specificity of specific gene target and further feature, and wherein siRNA has more specificity relatively for the particular target gene, and miRNA suppresses the translation of multiple mRNA material.
Attempted utilizing RNAi in treatment, to be used for the multiple disease patient's condition, like macular degeneration and respiratory syncytial virus infection (Sah, 2006, Life Sci 79:1773-80) through suppressing selected gene expression.Proposed that siRNA works in to the defence of the host cell of viral infection and with siRNA as the method for antiviral therapy carried out broad research (referring to for example Zhang etc., 2004, Nature Med 11:56-62; Novina etc., 2002, Nature Med 8:681-86; Palliser etc., 2006, Nature 439:89-94).Also attempt use siRNA and be used for treatment of cancer.Fujii etc. (2006, Int J Oncol 29:541-48) are used for to the positive cervical cancer cell of siRNA transfection HPV of HPV E6 and E7 and have suppressed tumor growth.Different mucin (metadherin) the expression blocking-up of siRNA mediation can suppress experimental lung metastasis (Brown and Ruoslahti, 2004, Cancer Cell 5:365-74) in the breast cancer cell according to reports.
Comprise the cell malabsorption of exogenous siRNA and to the potential effect of missing the target (referring to for example Kim etc., 2009, Trends Molec Med 15:491-500) of other gene based on the difficulty of the therapy of siRNA.Different siRNA and demonstration that Jackson etc. (2003, Nat Biotechnol 21:635-37) have designed targeting IGF-1R and MAPK14 contain the silence of lacking to the non-target gene of 11 continuous nucleotides identical with the siRNA material.
The targeted delivery of having attempted providing siRNA is with the reduction toxic probability of missing the target.Song etc. (2005, Nat Biotechnol 23:709-17) use the Fab fragment to the HIV envelope protein of coupling protamine that siRNA is delivered to circulating cells.Schiffelers etc. (2004, NuclAcids Res 32:e149) are coupled to nanoparticle with the RGD peptide and are delivered to tumor and have suppressed tumor-blood-vessel growth and the growth rate in the nude mouse will resist VEGF R2siRNA.Dickerson etc. use with the functionalized nanogel of anti-EphA2 receptor peptide to strengthen ovarian cancer cell to the chemosensitivity to the siRNA of EGFR.The magnetic nano-particle of coupling dendritic has been applied to targeted delivery antisense survivin oligodeoxynucleotide (Pan etc., 2007, Cancer Res 67:8156-63).
Although make progress recently, still the more effective means existence of targeted delivery therapeutic siRNA is needed in the art, absorb and make the toxic side effects relevant reduce to minimum (Kim etc., 2009) to increase cell with siRNA.
General introduction
The present invention relates to be used for the siRNA material effectively is delivered to the method and composition of disease association target cell, tissue or pathogen.Preferably, promote to send through using the targeted delivery complex, said targeted delivery complex comprises and the link coupled antibody of the carrier molecule of siRNA, antibody fragment or other targeted molecular, like protamine, dendritic or nanoparticle.More preferably, targeted molecular makes therapeutic siRNA high selectivity or is delivered to diseased cells or tissue specifically.More preferably, the targeted delivery complex promotes interior absorption of cell of siRNA in the target cell.
The siRNA material to be sent and the corresponding specificity of targeted molecular will be by disease patient's condition decisions to be treated.Discuss more in detail like hereinafter; 10/1000ths the siRNA sequence to the mRNA relevant with the extensive multiple disease patient's condition is well known in the art and can obtains from public database, as is positioned at the probe data storehouse (Probe database) that siRNAdb data base, the MIT/ICBP siRNA data base of bioinformatics center, Stockholm (Stockholm Bioinformatics Centre), the RNAi that is positioned at Boulder institute (Broad Institute) unite shRNA library (RNAiConsortium shRNA Library) and be positioned at NCBI.Many siRNA materials also can be buied from commercial supplier, as Sigma-Aldrich (St Louis, MO), Invitrogen (Carlsbad; CA), Santa Cruz Biotechnology (Santa Cruz, CA), Ambion (Austin, TX), Dharmacon (Thermo Scientific; Lafayette, CO), Promega (Madison, WI), Mirus Bio (Madison; WI) and Qiagen (Valencia, CA).
The existing report of the carrier part of the multiple siRNA of being used for and any said known carrier all can be incorporated the targeted delivery complex into for use.The limiting examples of carrier comprises protamine (Rossi, 2005, Nat Biotech 23:682-84; Song etc., 2005, Nat Biotech 23:709-17); Dendritic is like PAMAM dendritic (Pan etc., 2007, Cancer Res.67:8156-8163); PEI (Schiffelers etc., 2004, Nucl Acids Res 32:e149); Polypropylene imines (Taratula etc., 2009, J Control Release 140:284-93); Polylysine (Inoue etc., 2008, J Control Release 126:59-66); The reducible polycation (Stevenson etc., 2008, J Control Release 130:46-56) that contains histidine; Histone h1 albumen (Haberland etc., 2009, Mol Biol Rep 26:1083-93); Cationic comb copolymer (Sato etc., 2007, J Control Release 122:209-16); Polymer micelle (U.S. Patent Application Publication No.20100121043); And chitosan-thiamine pyrophosphate salt (Rojanarata etc., 2008, Pharm Res 25:2807-14).Those of skill in the art will recognize, in general, use polycation property protein or polymer as the siRNA carrier.Those of skill in the art will recognize further that the siRNA carrier also can be used for carrying other oligonucleotide or nucleic acid substances, like antisense oligonucleotide or short dna gene.
Various embodiments possibly relate to uses the targeted delivery complex to treat disease, includes but not limited to non-Hodgkin lymphomas (non-Hodgkin's lymphomas), the B cell is acute and chronic lymphatic leukemia, Burkitt lymphoma (Burkitt lymphoma), hodgkin's lymphomas, hairy cell leukemia, acute and chronic lymphocytic leukemia, t cell lymphoma and leukemia, multiple myeloma, glioma, macroglobulinemia Waldenstron (Waldenstrom's macroglobulinemia), carcinoma, melanoma, sarcoma, glioma and skin carcinoma.The group that the optional free oral cancer of carcinoma, gastrointestinal cancer, colon cancer, gastric cancer, lung tracheocarcinoma, pulmonary carcinoma, breast carcinoma, ovarian cancer, carcinoma of prostate, uterus carcinoma, carcinoma of endometrium, cervical cancer, bladder cancer, cancer of pancreas, osteocarcinoma, hepatocarcinoma, carcinoma of gallbladder, renal carcinoma, skin carcinoma and carcinoma of testis are formed.
In addition; Can use the targeted delivery complex to treat autoimmune disease, for example acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea (Sydenham's chorea), myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyadenous property syndrome, epidermolysis class sky bag skin ulcer, diabetes, Heng Nuoke-Si Qilaien purpura (Henoch-Schonlein purpura), post-streptococcal infection nephritis, erythema nodosum, high iS-One arteritis (Takayasu's arteritis), Addison's disease (Addison's disease), rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture syndrome (Goodpasture's syndrome), thromboangitis obliterans, Xiu Gelun syndrome (Sjogren's syndrome), primary biliary cirrhosis, struma lymphomatosa (Hashimoto's thyroiditis), thyroxine disease, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, Wei Genashi granulomatosis (Wegener's granulomatosis), membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis (tabes dorsalis), giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis or fibrosing alveolitis.
Compositions of the present invention also is applicable to the treatment infection, and wherein the antibody component specificity of targeted delivery complex combines pathogenic microorganism.In context of the present invention, pathogenic microorganism comprises pathogenic bacteria, virus, fungus and multiple parasite, but and these microorganisms of antibody targeting, its product or the antigen relevant with its pathological changes.The instance of microorganism includes but not limited to: streptococcus agalactiae (Streptococcus agalactiae); Legionella pneumophilia (Legionella pneumophilia); Streptococcus pyogenes (Streptococcus pyogenes); Escherichia coli (Escherichia coli); NEISSERIA GONORRHOEAE (Neisseria gonorrhoeae); Neisseria meningitidis (Neisseria meningitidis); Streptococcus pneumoniae (Pneumococcus); Hemophilus influenza B (Haemophilusinfluenzae B); Treponema pallidum (Treponema pallidum); Lyme disease spirochete (Lyme disease spirochetes); Pseudomonas aeruginosa (Pseudomonas aeruginosa); Mycobacterium leprae (Mycobacterium leprae); Alcaligenes abortus (Brucella abortus); Mycobacterium tuberculosis (Mycobacterium tuberculosis); Tetanus toxin (Tetanus toxin); HIV-1; HIV-2; HIV-3; Hepatitis A; Hepatitis B; Hepatitis C; Hepatitis D; Rabies virus; Influenza virus; Cytomegalovirus; Herpes simplex virus I and II; Human serum parvovirus appearance virus; Papillomavirus; Polyoma virus; Respiratory syncytial virus; Varicella zoster virus; Hepatitis B virus; Papillomavirus; Measles virus; Adenovirus; The HTLV; Epstein-Barr virus (Epstein-Barr virus); The muroid leucovirus; Mumps virus; Vesicular stomatitis virus; Sindbis alphavirus (Sindbis virus); The lymphatic choriomeningitis virus; The Verrucosis poison; Blue tongue virus (Blue tongue virus); Sendai virus (Sendai virus); Feline leukaemia virus; Reovirus; Poliovirus; Simian virus 40; Mouse mammary tumor virus; Dengue virus; Rubella virus; Protozoacide; Plasmodium falciparum (Plasmodium falciparum); Plasmodium vivax (Plasmodium vivax); Toxoplasma gondii (Toxoplasma gondii); The beautiful trypanosomicide in port (Trypanosoma rangeli); Schizotrypanum cruzi (Trypanosoma cruzi); Trypanosoma rhodesiense (Trypanosoma rhodesiense); Trypanosoma bocagei (Trypanosoma brucei); Schistosoma mansoni (Schistosoma mansoni); Schistosoma japonicum (Schistosoma japonicum); Babesia bovis (Babesia bovis); Eimeria tenella (Eimeria tenella); Onchocerca caecutiens (Onchocerca volvulus); Helcosoma tropicum (Leishmania tropica); Trichinella (Trichinella spiralis); Little Tai Leier piroplasm (Theileria parva); Taenia hydatigena (Taenia hydatigena); Taenia ovis (Taenia ovis); Taeniasis bovis (Taenia saginata); Echinococcus granulosus (Echinococcus granulosus); Mesocestoides corti (Mesocestoides corti); Mycoplasma arthritidis (Mycoplasma arthritidis); Mycoplasma hyorhinis (M.hyorhinis); Mycoplasma orale (M.orale); Mycoplasma arginini (M.arginini); Acholeplasma laidlawii (Acholeplasma laidlawii); Mycoplasma salivarium (M.salivarium) and mycoplasma pneumoniae (M.pneumoniae).Monoclonal antibody in conjunction with these pathogenic microbes is well known in the art.
In one embodiment, can use pharmaceutical composition of the present invention to treat to suffer from metabolic disease the experimenter of (like amyloidosis) or neurodegenerative disease (like the A Zihai Mo's disease).A crust pearl monoclonal antibody (Bapineuzumab) is in the clinical trial of A Zihai Mo's disease therapy.The antibody that other proposition is used to treat the A Zihai Mo's disease comprises Alz50 (Ksiezak-Reding etc., 1987, J Biol Chem 263:7943-47), sweet for Shandong monoclonal antibody (gantenerumab) and Suo Lan pearl monoclonal antibody (solanezumab).Report the sharp former times monoclonal antibody of anti-TNF-Alpha antibodies English (Infliximab) and reduced amyloid plaques and improvement cognition.Propose anti-cd 3 antibodies and be used to treat IDDM (Cernea etc., 2010, Diabetes Metab Rev 26:602-05).In addition, can use pharmaceutical composition of the present invention to treat the experimenter who suffers from the immune disorder disease, like graft versus host disease or organ transplant rejection.
In a preferred embodiment, can use the disease of the compositions advocated and method treatment to comprise cardiovascular disease, like fibrin clot, atherosclerosis, myocardial ischemia and infraction.Fibrin antibody (scFv (59D8) for example; T2G1s; MH1) be known and just carrying out clinical trial as the preparation that appears said grumeleuse and pulmonary infarction, and Anti granulocyte antibody (like MN-3, MN-15, anti-NCA95 and anti-CD15 antibody) but targeting myocardial infarction and myocardial ischemia.(referring to for example United States Patent(USP) No. 5,487,892; 5,632,968; 6,294,173; 7,541,440, the embodiment part of each patent is incorporated this paper by reference into).Can use anti-macrophage, anti-low density lipoprotein, LDL (LDL) and anti-CD74 (for example hLL1) antibody to come the targeting atherosclerotic plaque.Abciximab (Abciximab) (anti-glycoprotein iib/iiia) approved is got involved to intervene and treat to assist in the unstable angina pectoris at percutaneous coronary and is used for prevention of restenosis (Waldmann etc., 2000, Hematol 1:394-408).Report anti-cd 3 antibodies and reduced atherosclerotic generation and development (Steffens etc., 2006, Circulation 114:1977-84).In mouse model, induce the atherosclerotic improvement (Ginsberg, 2007, J Am Coll Cardiol 52:2319-21) of having set up to the antibody of oxidized ldl.Shown the ischemic cell injury (Zhang etc., 1994, Neurology 44:1747-51) anti-ICAM-1 antibody reduces cerebral artery occlusion in rat after.By the anti-T cell monoclonal of following representative: OKT antibody (can available from Ortho Pharmaceutical Company), it combines normal T lymphocyte to the commercially available monoclonal of HLA; Monoclonal antibody by hybridoma generation with ATCC deposit number HB44, HB55, HB12, HB78 and HB2; G7E11, W8E7, NKP15 and GO22 (Becton Dickinson); NEN9.4 (New EnglandNuclear); And FMC11 (Sera Labs).Description to the antibody of fibrin and platelet antigen is contained in Knight, and Semin.Nucl.Med. is among the 20:52-67 (1990).
Employed antibody or other targeted molecular can combine any disease association antigen as known in the art.For instance; In the disease patient's condition is under the situation of cancer; Be well known in the art by tumor cells expression or otherwise relevant many antigens with tumor cell; Include but not limited to: carbonic anhydrase IX, α-fetoprotein, α-actinine-4, A3, to the specific antigen of A33 antibody tool, ART-4, B7, Ba 733, BAGE, BrE3 antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CXCR4, colon-specific antigen p (CSAp), CEA (CEACAM5), CEACAM6, c-met, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3, folacin receptor, G250 antigen, GAGE, gp100, GROB, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunit, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, insulin-like growth factor-i (IGF-1), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR/FUT, MIF (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, to the specific antigen of PAM-4 antibody tool, placental growth factor, p53, PLAGL2, PAP, PSA, PRAME, PSMA, P1GF, IGF, IGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-α, Tn antigen, thomson-Friedrich antigen (Thomson-Friedenreich antigens), neoplasm necrosis antigen, VEGFR, ED-B fibronectin, WT-1,17-1A antigen, complement factor C3, C3a, C3b, C5a, C5, angiogenesis sign, bcl-2, bcl-6, Kras, cMET, oncogene sign and oncogene product (referring to for example Sensi etc.; Clin Cancer Res 2006,12:5023-32; Parmiani etc., J Immunol 2007,178:1975-79; CancerImmunol Immunother 2005 such as Novellino, 54:187-207).
Available exemplary antibodies includes but not limited to: hR1 (anti-IGF-1R, the U.S. Patent application No.12/722 of 3/12/10 submission, 645), hPAM4 (anti-stick albumen, United States Patent(USP) No. 7,282,567), hA20 (anti-CD20; United States Patent(USP) No. 7,251,164), hA19 (anti-CD19, United States Patent(USP) No. 7,109,304), hIMMU31 (anti-AFP; United States Patent(USP) No. 7,300,655), hLL1 (anti-CD74, United States Patent(USP) No. 7,312,318), hLL2 (anti-CD22; United States Patent(USP) No. 7,074,403), hMu-9 (anti-CSAp, United States Patent(USP) No. 7,387,773), hL243 (anti-HLA-DR; United States Patent(USP) No. 7,612,180), hMN-14 (anti-CEACAM5, United States Patent(USP) No. 6,676,924), hMN-15 (anti-CEACAM6; United States Patent(USP) No. 7,541,440), hRS7 (anti-EGP-1, United States Patent(USP) No. 7,238; 785) and hMN-3 (anti-CEACAM6, U.S. Patent application No.7,541,440), the embodiment of each referenced patents or application part is incorporated this paper by reference into.
Employed antibody or Fab can be chimeric, humanization or people.For parent's rodent antibody, preferably use chimeric antibody, because it has people's antibody constant region sequence and therefore can not cause with the equally strong human anti-mouse antibody of rodent antibody (HAMA) and react.Even more preferably use humanized antibody, so that further reduce the probability of bringing out the HAMA reaction.Such as hereinafter argumentation, through muroid framework region and constant region sequence are replaced into corresponding human antibody framework district and constant region sequence to rodent antibody carry out humanized technology be in the art know and be applied to numerous muroid anticancrins.The antibody humanization also possibly relate to one or more people's framework amino acid residues are substituted by the corresponding residue from parent's muroid framework region sequence.Equally such as hereinafter argumentation, the technology of manufacturer's antibody is also known.
In some preferred embodiment, the targeted delivery complex can use butt joint locking (DNL) technology to make (referring to for example United States Patent(USP) No. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400, the embodiment part of each patent is incorporated this paper by reference into).The DNL techniques make use is from the dimerization of PKA and docking structure territory (DDD part) and from the specificity binding interactions that takes place between any anchoring structure territory (AD part) in the multiple known A kinases anchorin (AKAP).The DDD part spontaneously forms dimer, and it then combines the AD part.Through suitable effect part (like antibody or its fragment and siRNA carrier) is connected in AD and DDD part, the DNL technology allows the specificity covalency to form any desired targeted delivery complex.Be divided at effector under the situation of protein or peptide (like antibody fragment, protamine or polylysine), AD and DDD part can incorporate into the link coupled fusion rotein of effect part in.Under the situation of utilizing nonprotein effect part, chemical coupling capable of using connects AD or DDD part.
In certain embodiments, the physics of implementing through targeted delivery siRNA can strengthen through the combination treatment with one or more other therapeutic agents.Employed known treatment agent comprises toxin, immunomodulator (like cytokine, lymphokine, chemotactic factor, somatomedin and tumor necrosis factor), hormone, hormone antagonist, enzyme, oligonucleotide (like siRNA or RNAi), photosensitive therapeutic agent, anti-angiogenic agent and short apoptosis agent.Therapeutic agent can be through with identical or different antibody or other targeted molecular coupling is sent or not coupling form is sent.Other therapeutic agent can be before siRNA targeted delivery complex, simultaneously or use afterwards.
In a preferred embodiment, therapeutic agent is a cytotoxic agent, like medicine or toxin.Equally preferably, medicine is selected from the group of being made up of following: chlormethine, aziridine derivant, alkylsulfonate, nitroso ureas, gemcitabine (gemcitabine), triazenes, folacin, anthracycline, taxane, cox 2 inhibitor, pyrimidine analogue, purine analogue, antibiotic, enzyme inhibitor, epipodophyllotoxin, platinum coordination complex, vinca alkaloids, replacement urea, methyl hydrazine derivant, adrenal cortex inhibitor, hormone antagonist, Endostatin, taxol, camptothecine, SN-38, amycin (doxorubicins) and its analog, antimetabolite, alkylating agent, antimitotic agent, anti-angiogenic agent, tyrosine kinase inhibitor, mTOR inhibitor, heat shock protein (HSP90) inhibitor, proteasome inhibitor, hdac inhibitor, short apoptosis agent, methotrexate, CPT-11, calicheamicin (calicheamicin) and its combination.
In another preferred embodiment, therapeutic agent is the toxin that is selected from by the following group of forming: Ricin (ricin), abrin (abrin), alpha toxin (alpha toxin), saporin (saporin), ribonuclease (RNA enzyme), DNA enzyme I, staphyloentero-toxin-A (Staphylococcal enterotoxin-A), PAP (pokeweed antiviral protein), gelonin (gelonin), diphtheria toxin, diphtherotoxin (diphtheria toxin), PE (Pseudomonas exotoxin) and pseudomonas endotoxin (Pseudomonas endotoxin) with and the combination.Perhaps be selected from immunomodulator: cytokine, stem cell factor, lymphotoxin, Hemopoietic factor, group's stimulating factor (CSF), interferon (IFN), erythropoietin, thrombopoietin and its combination by the following group of forming.
In other preferred embodiment, therapeutic agent is the radionuclide that is selected from by the following group of forming: 111In, 177Lu, 212Bi, 213Bi, 211At, 62Cu, 67Cu, 90Y, 125I, 131I, 32P, 33P, 47Sc, 111Ag, 67Ga, 142Pr, 153Sm, 161Tb, 166Dy, 166Ho, 186Re, 188Re, 189Re, 212Pb, 223Ra, 225Ac, 59Fe, 75Se, 77As, 89Sr, 99Mo, 105Rh, 109Pd, 143Pr, 149Pm, 169Er, 194Ir, 198Au, 199Au with 211Pb.The radionuclide that decays in fact with the Auger emitted particle also is preferred.For example Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161, Os-189m and Ir-192.The decay that is suitable for beta-particle emission nucleic can be preferably 1,000keV, more preferably < 100keV, and most preferably be < 70keV.The radionuclide of decay also is preferred in fact along with the alpha-particle generation.Said radionuclide includes but not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255.The decay that is suitable for alpha-particle emission radionuclide can be preferably 2,000-10, and 000keV, more preferably 3,000-8,000keV, and most preferably be 4,000-7,000keV.Other potential suitable radiosiotope comprises 11C, 13N, 15O, 75Br, 198Au, 224Ac, 126I, 133I, 77Br, 113MIn, 95Ru, 97Ru, 103Ru, 105Ru, 107Hg, 203Hg, 121MTe, 122MTe, 125MTe, 165Tm, 167Tm, 168Tm, 197Pt, 109Pd, 105Rh, 142Pr, 143Pr, 161Tb, 166Ho, 199Au, 57Co, 58Co, 51Cr, 59Fe, 75Se, 201Tl, 225Ac, 76Br, 169Yb etc.In other embodiments, therapeutic agent is the photosensitive therapeutic agent that is selected from the group of being made up of chromogen and dyestuff.
Perhaps, therapeutic agent is the enzyme that is selected from by the following group of forming: malic dehydrogenase, staphylococcal nuclease, δ-V-steroid isomerase, Alcohol Dehydrogenase from Yeast, α-Gan Youlinsuantuoqingmei, triose-phosphate isomerase, horseradish peroxidase, alkali phosphatase, asparaginase, glucoseoxidase, beta galactosidase, ribonuclease, urase, catalase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and Acetylcholinesterase.Said enzyme can with for example become the prodrug combination of cytotoxic agent to use by enzymatic conversion with nontoxic relatively administered and at target site.In other replacement scheme, medicine can change into the low form of toxicity by endogenous enzyme in subject, but possibly change into the cytotoxicity form again by the therapeutic enzyme.
The accompanying drawing summary
Fig. 1. be used for the synthetic of E1-L-thP1DNL complex that siRNA sends.(A) manufacturing has the terminal anti-Trop-2hRS7 antibody of C-that AD2 partly is connected in each heavy chain of antibody.(B) synthetic DDD2-L-thP 1 construct with the truncate section of people's protamine 1 and N-end terminal with the C-that DDD2 partly is connected to the humanized antibody light chain.(C) under the week reduction condition, hatch the hRS7DNL complex that hRS7-IgG-AD2 and DDD2-L-thP 1 cause forming the coupling protamine, called after E1-L-thP1.
The bonded gel shift of the DNA of Fig. 2 .E1-L-thP1 is measured.
Fig. 3. combine the internalization of the siRNA of mediation by E1-L-thP1.
Fig. 4. by the inductive apoptosis of E1-L-thP1 internalization of siRNA.
Describe in detail
Definition
Unless other regulation, one (" a/an) mean one (kind) or a plurality of (kinds).”
As used herein, " pact " means and adds deduct 10%.For example, " 100 " will comprise any numerical value between 90 and 110 approximately.
As described herein; Antibody is meant that total length (promptly; Naturally occurring or form through the immunoglobulin gene fragment recombination method) immunocompetence (that is, the specificity combines) part of immunoglobulin molecules (for example IgG antibody) or immunoglobulin molecules, like antibody fragment.
Antibody fragment is the part of antibody, like F (ab') 2, Fab', Fab, Fv, sFv etc.With structure-irrelevant, antibody fragment combines with the same antigen of being discerned by full length antibody.Term " antibody fragment " also comprises the isolated fragment of being made up of the variable region of antibody, like " Fv " fragment of forming by the variable region of heavy chain and light chain, and the recombinant single chain peptide molecule (" scFv albumen ") that is connected by the peptide connexon with heavy chain and variable region of light chain.
Chimeric antibody is a kind of recombiant protein, and it contains the antibody that derives from species, and the variable domains of preferred rodent animal antibody comprise complementary determining region (CDRs), and the constant domain of antibody molecule is the constant domain that derives from people's antibody.For veterinary applications, the constant domain of chimeric antibody can derive from other species, like the constant domain of cat or Canis familiaris L..
Humanized antibody is a kind of recombiant protein, wherein will be from the antibody of species, and for example the CDRs of rodent animal antibody transfers to people's heavy chain and the light chain variable domain (for example framework region sequence) from the variable heavy chain and the variable light chain of rodent animal antibody.The constant domain of antibody molecule is the constant domain that derives from people's antibody.In certain embodiments, can the framework region amino acid residue of parent (rodent) antibody of limited quantity be replaced adult's antibody framework region sequence.
People's antibody is for example from carrying out the antibody that " engineered " obtains with the transgenic mice that produces persona certa's antibody in response to antigen stimulation.In this technology, the element of people's heavy chain and light chain gene seat to be introduced in the mouse species that is obtained by embryonic stem cell line, the targeting that said strain contains endogenous muroid heavy chain and light chain gene seat destroys.Transgenic mice can synthesize the specific people's antibody of specific antigen tool, and said mice can be used for producing the hybridoma of secretion people antibody.The method that is used for obtaining people's antibody from transgenic mice is by Green etc., Nature Genet.7:13 (1994); Lonberg etc., Nature 368:856 (1994); With Taylor etc., Int.Immun.6:579 (1994) describes.Also can be through heredity or chromosome transfection method and display technique of bacteriophage structure fully human antibodies, said method and technology all are known in the art.Referring to for example McCafferty etc., Nature 348:552-553 (1990), it is described from from external generations of immunoglobulin variable domain gene pedigree people antibody and its fragment of epidemic disease donor rather.In this technology, the antibody variable domain gene is cloned in the main or less important coat protein gene of filobactivirus with frame, and on the surface of bacteriophage particles, is shown as the functional antibodies fragment.Because thread particle contains the single stranded DNA copy of phage genome, so select the gene of the antibody that also causing selects to encode presents said character based on the functional character of antibody.In this way, some character of phage simulation B cell.Phage display can be carried out by multiple form, about summary, sees also for example Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).People's antibody also can be produced by external activating B cell.Referring to United States Patent(USP) No. 5,567,610 and 5,229,275, the embodiment part is incorporated this paper by reference into.
Therapeutic agent is to separate with antibody moiety, simultaneously or use in regular turn or with antibody moiety (that is, antibody or antibody fragment, or sub-fragment) coupling and be applicable to chemical compound, molecule or the atom of treatment disease.The instance of therapeutic agent comprises antibody, antibody fragment, medicine, toxin, nuclease, hormone, immunomodulator, short apoptosis agent, anti-angiogenic agent, boron compound, photosensitizer or dyestuff and radiosiotope.Employed therapeutic agent describes in further detail hereinafter.
Immune conjugate is and at least a therapeutic agent and/or the link coupled antibody of diagnostic agent, antibody fragment or fusion rotein.
RNA interfering
Those of skill in the art will recognize that any siRNA or RNA interfering material all can be connected to the targeted delivery complex for being delivered to target tissue.Many siRNA materials to multiple target are well known in the art, and in the method and composition of being advocated any said known siRNA capable of using.
The known siRNA material that might use comprises following person: the IKK-γ (United States Patent (USP) 7,022,828) that has the specificity; VEGF, Flt-1 and Flk-l/KDR (United States Patent (USP) 7,148,342); Bcl2 and EGFR (United States Patent (USP) 7,541,453); CDC20 (United States Patent (USP) 7,550,572); Transducin (β) appearance albumen 3 (United States Patent (USP) 7,576,196); KRAS (United States Patent (USP) 7,576,197); Carbonic anhydrase II (United States Patent (USP) 7,579,457); Complement component 3 (United States Patent (USP) 7,582,746); Interleukin-1 receptor associated kinase 4 (IRAK4) (United States Patent (USP) 7,592,443); Survivin (United States Patent (USP) 7,608,7070); Superoxide dismutase 1 (United States Patent (USP) 7,632,938); MET proto-oncogene (United States Patent (USP) 7,632,939); Amyloid beta presoma albumen (APP) (United States Patent (USP) 7,635,771); IGF-1R (United States Patent (USP) 7,638,621); ICAM1 (United States Patent (USP) 7,642,349); Complement factor B (United States Patent (USP) 7,696,344); P53 (7,781,575) and apolipoprotein B (7,795,421), the embodiment part of each referenced patent is incorporated this paper by reference into.
Other siRNA material can obtain from known commercial source, as Sigma-Aldrich (St Louis, MO), Invitrogen (Carlsbad; CA), Santa Cruz Biotechnology (Santa Cruz, CA), Ambion (Austin, TX), Dharmacon (Thermo Scientific; Lafayette, CO), Promega (Madison, WI), Mirus Bio (Madison; WI) and Qiagen (Valencia, CA) and many other sources.The source that other of siRNA material can openly obtain comprises the siRNAdb data base, the MIT/ICBP siRNA data base that are positioned at bioinformatics center, Stockholm, is positioned at the probe data storehouse of winning graduate RNAi associating shRNA library and being positioned at NCBI.For example, in NCBI probe data storehouse, there are 30,852 kinds of siRNA materials.Those of skill in the art will recognize that for any target gene, perhaps the siRNA material designs, and perhaps it can easily use the software tool that can openly obtain to design.Any said siRNA material all can use target DNL complex to send.
Exemplary siRNA material as known in the art is listed in the table 1.Although siRNA sends as duplex molecule, the reason from simplicity only illustrates the sense strand sequence in the table 1.
The exemplary siRNA sequence of table 1.
Figure BDA00001666436900151
Figure BDA00001666436900161
Those of skill in the art will recognize, the minute quantity sample in the table 1 oblatio siRNA material as known in the art sum, and in any said known siRNA method and composition of all can be used for being advocated.
Antibody Preparation
In certain embodiments, the targeted delivery complex can comprise monoclonal antibody (MAb) or antigen binding antibody fragment.MAb can separate and purification from the hybridoma culture through multiple technology of having established.Said isolation technics comprises a-protein or protein G agarose affinity chromatography, size exclusion chromatography and ion exchange chromatography.2.7.1-2.7.12 page or leaf and 2.9.1-2.9.3 page or leaf referring to for example Coligan.In addition referring to Baines etc., " Purification of Immunoglobulin G (IgG), " METHOD S IN MOLECULAR BIOLOGY, the 10th volume, the 79-104 page or leaf (The Humana Press, Inc.1992).Initially produced to after the immunogenic antibody, but antagonist checks order and prepares through recombinant technique subsequently.The humanization of rodent antibody and antibody fragment and chimeric is known by those skilled in the art, such as hereinafter argumentation.
Chimeric antibody
Chimeric antibody is a kind of recombinant antibodies, and wherein the variable region of people's antibody has been replaced into the for example variable region of mouse antibodies, comprises the complementary determining region (CDR) of mouse antibodies.Chimeric antibody shows immunogenicity and the enhanced stability that reduces when being applied to the experimenter.The current techique of clone's muroid immunoglobulin variable domain for example is disclosed in Orlandi etc., among the Proc.Nat ' l Acad.Sci.USA 6:3833 (1989).The technology that makes up chimeric antibody is known by those skilled in the art.For instance, Leung etc., Hybridoma 13:469 (1994) is through the V of the anti-CD22 antibody of the muroid LL2 that will encode κAnd V HDomain and corresponding human κ and IgG 1The DNA sequence combination results of constant region domain the LL2 chimera.
Humanized antibody
The technology that produces humanization MAb be in the art know (referring to for example Jones etc., Nature 321:522 (1986); Riechmann etc., Nature 332:323 (1988); Verhoeyen etc., Science 239:1534 (1988); Carter etc., Proc.Nat ' l Acad.Sci.USA 89:4285 (1992); Sandhu, Crit.Rev.Biotech.12:437 (1992); With Singer etc., J.Immun.150:2844 (1993)).Chimeric or muroid monoclonal antibody can be through carrying out humanization in the corresponding variable domains that will transfer to people's antibody from the mice CDR of the variable heavy chain of mouse immuning ball protein and light chain.Mice framework region (FR) in the chimeric mAb is also replaceable to be people FR sequence.Because simply with mice CDR transfer to cause affinity of antibody to reduce among the people FR usually or even forfeiture, so possibly carry out extra modification to recover the initial affinity of rodent antibody.This measure can realize the antibody that its epi-position has the good combination affinity with acquisition through the one or more people's residues in the FR district being replaced into its muroid homologue.Referring to for example Tempest etc., Biotechnology 9:266 (1991) and Verhoeyen etc., Science 239:1534 (1988).Be used for substituted preferred residue comprise 1,2 or 3 dusts that are positioned at CDR residue side chain with, be positioned near the CDR sequence or prediction and the interactional FR residue of CDR residue.
People's antibody
Use combined method or be well known in the art (Mancini etc. for example, 2004, New Microbiol 27:315-28 through the method that the transgenic animal that human immunoglobulin gene's seat transforms produce fully human antibodies; Conrad and Scheller, 2005, Comb.Chem.High Throughput Screen.8:117-26; Brekke and Loset, 2003, Curr.Opin.Pharmacol.3:544-50).Also can be through heredity or chromosome transfection method and display technique of bacteriophage structure fully human antibodies, said method and technology all are known in the art.Referring to for example McCafferty etc., Nature 348:552-553 (1990).Expect that the performance of said fully human antibodies is than chimeric or humanized antibody even side effect still less and play endogenous people antibody basically in vivo.
In a replacement scheme, can use display technique of bacteriophage produce people's antibody (Dantas-Barbosa etc. for example, 2005, Genet.Mol.Res.4:126-40).People's antibody can produce (Dantas-Barbosa etc., 2005) by the normal person or by the people who shows the specified disease patient's condition (like cancer).The advantage that makes up people's antibody by diseased individuals is that the circulating antibody pedigree possibly be partial to the antigenic antibody of disease association.
In a limiting examples of the method, Dantas-Barbosa etc. (2005) have been made up the phage display library of human Fab's antibody fragment by the osteosarcoma patient.In general, obtain total RNA (the same) from the blood circulation lymphocyte.Clone reorganization Fab and insert (the same) the phage display library from μ, γ and κ chain antibody pedigree.RNA is transformed into cDNA and is used to use the Auele Specific Primer to heavy chain and light chain immunoglobulin sequences to prepare Fab cDNA library (Marks etc., 1991, J Mol.Biol.222:581-97).Library construction is to carry out (2000:Phage Display Laboratory Manual according to Andris-Widhopf etc.; Barbas etc. (writing), the 1st edition, Cold Spring Harbor Laboratory Press; ColdSpring Harbor, the 9.1st to 9.22 page of NY).Final Fab fragment digests with restriction endonuclease and inserts in the phage genome with the preparation phage display library.Can through standard phage display method said library be screened as known in the art.Phage display can be carried out by multiple form, about its summary, sees also for example Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).
People's antibody also can be produced by external activating B cell.Referring to United States Patent(USP) No. 5,567,610 and 5,229,275, its by reference integral body incorporate this paper into.Those of skill in the art will recognize, these technology are as an example and any known method of preparation capable of using and screening people's antibody or antibody fragment.
In another replacement scheme, can use and carry out genetically engineeredly using the standard immunoassay scheme to produce to the antibody of any immunogenicity target basically with the transgenic animal that produce people's antibody.The method that is used for obtaining people's antibody from transgenic mice is by Green etc., Nature Genet.7:13 (1994); Lonberg etc., Nature 368:856 (1994); With Taylor etc., Int.Immun.6:579 (1994) is open.The limiting examples of said system is from Abgenix (Fremont; CA)
Figure BDA00001666436900191
(Green etc. for example; 1999; J.Immunol.Methods 231:11-23 incorporates this paper by reference into).In
Figure BDA00001666436900192
and similar animal; Made the mouse antibodies gene inactivation and be replaced into the functional human antibody gene, and the remainder of mouse immune system is kept perfectly.
with containing groups of people IgH and Ig kappa gene seat, comprised that most of variable region sequences transforms together with the system genitale configuration YACs (yeast artificial chromosome) of auxiliary gene and regulating and controlling sequence.But end user variable region pedigree produces antibody and produces the B cell, and it can be processed into hybridoma through known technology.
Figure BDA00001666436900194
with the target antigen immunity will produce people's antibody through normal immunoreaction, and it can collect and/or make through the standard technique that preceding text are discussed.Can obtain the multiple strain of
Figure BDA00001666436900201
, it can produce different classes of antibody separately.Proved that people's antibody that transgenic produces has treatment potential, keeps the pharmacokinetic property (Green etc., 1999) of normal person's antibody simultaneously.Those of skill in the art will recognize; Compositions of being advocated and method are not limited to use
Figure BDA00001666436900202
system, but any transgenic animal that carried out genetically engineered with generation people antibody capable of using.
Known antibody and target antigen
Such as preceding text argumentation; In preferred embodiments, the targeted delivery complex is to be used to treat malignant disease, cardiovascular disease, infectious disease, diseases associated with inflammation, autoimmune disease, metabolic disease, immune dysfunction disease (for example graft versus host disease or organ transplant rejection) or neural (for example neurodegenerative) disease.The exemplary target antigen that is used to treat said disease comprises carbonic anhydrase IX; CCCL19; CCCL21; CSAp; CD1; CD1a; CD2; CD3; CD4; CD5; CD8; CD11A; CD14; CD15; CD16; CD18; CD19; IGF-1R; CD20; CD21; CD22; CD23; CD25; CD29; CD30; CD32b; CD33; CD37; CD38; CD40; CD40L; CD45; CD46; CD52; CD54; CD55; CD59; CD64; CD66a-e; CD67; CD70; CD74; CD79a; CD80; CD83; CD95; CD126; CD133; CD138; CD147; CD154; CXCR4; CXCR7; CXCL12; HIF-1 α; AFP; PSMA; CEACAM5; CEACAM6; C-met; B7; The ED-B of fibronectin; Factor H; FHL-1; Flt-3; Folacin receptor; GROB; HMGB-1; Hypoxia inducible factor (HIF); HM1.24; Insulin-like growth factor-i (ILGF-1); IFN-γ; IFN-α; IFN-β; IL-2; IL-4R; IL-6R; IL-13R; IL-15R; IL-17R; IL-18R; IL-6; IL-8; IL-12; IL-15; IL-17; IL-18; IL-25; IP-10; MAGE; MCRP; MCP-1; MIP-1A; MIP-1B; MIF; MUC1; MUC2; MUC3; MUC4; MUC5; NCA-95; NCA-90; Ia; HM1.24; EGP-1; EGP-2; HLA-DR; Tenascin; Le (y); RANTES; T101; TAC; Tn antigen; Thomson-Friedrich antigen; Neoplasm necrosis antigen; TNF-α; TRAIL receptor (R1 and R2); VEGFR; EGFR; P1GF; Complement factor C3; C3a; C3b; C5a; C5; PLAGL2 and oncogene product.
In certain embodiments, like the treatment tumor, but employed antibody target tumor related antigen.These antigenicity signs can be the material that tumor produces or can be at tumor locus, on tumor cell surface or at the material of tumor cell inner accumulation.Said tumor correlating markings part is by following discloses: Herberman; " Immunodiagnosis of Cancer ", Fleisher writes, " The Clinical Biochemistry of Cancer "; The 347th page of (association (American Association of Clinical Chemists) of U.S. clinical chemistry man; 1979) and United States Patent(USP) No. 4,150,149; 4,361,544; With 4,444,744, the embodiment of said each document part is incorporated this paper by reference into.Report about tumor associated antigen (TAAs) comprises Mizukami etc., (2005, Nature Med.11:992-97); Hatfield etc., (2005, Curr.Cancer Drug Targets 5:229-48); Vallbohmer etc. (2005, J Clin.Oncol.23:3536-44); With Ren etc. (2005, Ann.Surg.242:55-63), incorporate this paper by reference into about each document of the TAA that differentiated.
The tumor correlating markings is by Herberman, and the same ranging in the plurality of classes comprises carcinoembryonic antigen, pregniotin, carcinogenic or oncovirus related antigen, organizes related antigen, organ related antigen, ectopic hormone and normal antigen or its variant.Sometimes, advantageously use the subunit of tumor correlating markings to produce antibody, for example the γ district of the β subunit of human chorionic gonadotropin (HCG) or carcinoembryonic antigen (CEA) with higher tumour-specific; It stimulates and the significantly reduced antibody generation of the cross reactivity of non-tumor material; Like United States Patent(USP) No. 4,361,644 and 4; Disclosed in 444,744.
Another blip is to stride the film activation factor and the mutual factor of CAML-(TACI).Referring to Nat.Immunol.1:252-256 such as Yu (2000).Briefly, TACI is the sign of B cell malignant disease (for example lymphoma).TACI and B cell maturation antigen (BCMA) are combined by tumor necrosis factor congener proliferation-inducing ligand (APRIL).APRIL stimulates elementary B cell and T cells in vitro to breed and because of accumulation B cell spleen weight is increased in vivo.APRIL also competes receptors bind with TALL-I (being also referred to as BLyS or BAFF).Solubility BCMA and TACI specificity stop the combination of APRIL and block the propagation of the APRIL stimulation of elementary B cell.BCMA-Fc also suppresses the production of antibodies to key hole spiral shell hemocyanin (keyhole limpet hemocyanin) and pneumovax (Pneumovax) in the mice body, show that the APRIL and/or the TALL-I signal transduction that carry out through BCMA and/or TACI are required for producing humoral immunization.Therefore, APRIL-TALL-I and BCMA-TACI form the two parts-amboceptor path of participating in stimulating B cell and T cell function.
Comprise under the situation of lymphoma, leukemia or autoimmune disease the group of forming below the optional freedom of target antigen: CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD67, CD74, CD79a, CD80, CD126, CD138, CD154, CXCR4, B7, MUC1, Ia, Ii, HM1.24, HLA-DR, tenascin, VEGF, P1GF, ED-B fibronectin, oncogene (for example c-met or PLAGL2), oncogene product, CD66a-d, downright bad antigen, IL-2, T101, TAG, IL-6, MIF, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) in disease.
Those of skill in the art will recognize, and will be capable of using as known in the art to having any antibody or the fragment of binding specificity with the disease patient's condition or the relevant target antigen of condition of illness.Said known antibodies includes but not limited to: hR1 (anti-IGF-1R, the U.S. Patent application No.12/772 of 3/12/10 submission, 645), hPAM4 (anti-cancer of pancreas mucin, United States Patent(USP) No. 7,282,567), hA20 (anti-CD20; United States Patent(USP) No. 7,251,164), hA19 (anti-CD19, United States Patent(USP) No. 7,109,304), hIMMU31 (anti-AFP; United States Patent(USP) No. 7,300,655), hLL1 (anti-CD74, United States Patent(USP) No. 7,312,318), hLL2 (anti-CD22; United States Patent(USP) No. 7,074,403), hMu-9 (anti-CSAp, United States Patent(USP) No. 7,387,773), hL243 (anti-HLA-DR; United States Patent(USP) No. 7,612,180), hMN-14 (anti-CEACAM5, United States Patent(USP) No. 6,676,924), hMN-15 (anti-CEACAM6; United States Patent(USP) No. 7,662,378, the 7/29/10 U.S. Patent application No.12/846 that submit to, 062), hRS7 (anti-EGP-1; United States Patent(USP) No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Patent application No.7,541; 440), Ab124 and Ab125 (anti-CXCR4, United States Patent(USP) No. 7,138,496), the embodiment part of each referenced patents or application is incorporated this paper by reference into.
Employed various other antibody be well known in the art (for example United States Patent(USP) No. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491; 7,612,180; 7,642,239 with U.S. Patent Application Publication No.20060193865; Incorporate this paper separately by reference into.)
Employed antibody can be buied from multiple known source.For example, the hybridoma cell line of multiple secretory antibody can be from American type culture collection (American Type Culture Collection; ATCC, Manassas VA) obtains.A large amount of antibody to various diseases target (including but not limited to tumor associated antigen) have been preserved in ATCC and/or have announced variable region sequences and the method and composition that can obtain to be advocated to be used for.Referring to for example United States Patent(USP) No. 7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803; 7,041,082; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,15; 6,716,966; 6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040; 6,451,310; 6,444,206; 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953; 5,525,338.It is merely exemplary and multiple other antibody is well known in the art with its hybridoma.Those of skill in the art will recognize, can pass through in simple search ATCC, NCBI and/or the USPTO information bank antibody to the relevant target of selected target disease and obtain to almost antigenic antibody sequence of any disease association or antibody-secreting hybridoma.The antigen binding structural domain of institute's clonal antibody can use that the standard technique of knowing in this area increases, excises, is connected in the expression vector, transfection is used for protein and produces to adapting to the host cell neutralization.
Antibody fragment
Antibody fragment is the antigen-binding portion thereof of antibody, like F (ab') 2, Fab', F (ab) 2, Fab, Fv, sFv, scFv etc.Can produce the antibody fragment of identification defined epitope through known technology.F (ab') 2Fragment for example can be through producing with the pepsin digested antibody molecule.These with other method by Goldenberg, United States Patent(USP) No. 4,036,945 and 4,331,647 and the description of wherein contained list of references.Also referring to Nisonoff etc., Arch Biochem.Biophys.89:230 (1960); Porter, Biochem.J.73:119 (1959); Edelman etc., METHODS INENZYMOLOGY the 1st volume, the 422nd page (Academic Press 1967); With Coligan 2.8.1-2.8.10 page or leaf and 2.10.-2.10.4 page or leaf.Perhaps, can make up the Fab' expression library (Huse etc., 1989, Science is 246:1274-1281) to allow fast and easily to differentiate the having specific monoclonal Fab' fragment of expectation.
Strand Fv molecule (scFv) comprises VL domain and VH domain.VL and VH domain associate and form the target binding site.These two domains are further covalently bound by peptide connexon (L).The scFv molecule is to be expressed as VL-L-VH under the situation of N end parts of scFv molecule at the VL domain, perhaps under the VH domain is the situation of N end parts of scFv molecule, is expressed as VH-L-VL.Preparing the scFv molecule is described in the following document with the method that design is fit to the peptide connexon: United States Patent(USP) No. 4,704,692; United States Patent(USP) No. 4,946,778; R.Raag and M.Whitlow, " Single Chain Fvs. " FASEB volume: 973-80 (1995) and R.E.Bird and B.W.Walker, " Single Chain Antibody Variable Regions, " TIBTECH, the 9th volume: 132-137 (1991).
Other antibody fragment, single domain antibody fragment for example, be well known in the art and the construct that can be used for being advocated in.Single domain antibody (VHH) can obtain from for example camel, alpaca or yamma through the standard immunoassay technology.(referring to for example Muyldermans etc., TIBS26:230-235,2001; Yau etc., J Immunol Methods 281:161-75,2003; Maass etc., J Immunol Methods 324:13-25,2007).VHH can have effective antigen binding capacity and can interact to novel epi-position that can not be approaching with conventional VH-VL.(Muyldermans etc., 2001).The alpaca serum IgG contain have an appointment 50% heavy chain only arranged camel IgG antibody (HCAb) (Maass etc., 2007).Alpaca can be used in known antigens (like TNF-α) immunity and the separable combination also and the VHH (Maass etc., 2007) of target antigen.Identified the PCR primer of the nearly all alpaca VHH coded sequence of amplification and can be used for making up alpaca VHH phage display library, said library can be used for the standard biological panning technique separation antibody fragment (Maass etc., 2007) through knowing in this area.
Antibody fragment also can be through carrying out Proteolytic enzyme or preparing through in escherichia coli or another host, expressing the said segmental DNA of coding to full length antibody.Antibody fragment can obtain with pepsin or papain digestion full length antibody through conventional method.For example, antibody fragment can be called F (ab') through producing so that the fragment of about 100Kd to be provided with pepsin enzymatic lysis antibody 2This fragment can be used thiol reductant and the blocking group of the sulfydryl that randomly is used for being produced by the disulfide bond cracking carries out further cracking to produce the Fab' unit price fragment of about 50Kd.
Perhaps, use papain to carry out enzymatic lysis and directly produce two unit price Fab fragments and a Fc fragment.
Also can use other method of cracking antibody, as separate heavy chain with form unit price light-heavy chain fragment, further crack fragment, or other enzymatic, chemistry or genetic technique are as long as said fragment combines the antigen by complete antibody identification.
The general technology that is used for antibody cloning and manufacturing
Various technology, as make chimeric or humanized antibody, possibly relate to the program of antibody cloning and structure.The antigen of target antibody combines V κ(variable light chain) and V H(variable heavy chain) sequence can obtain through multiple molecular cloning program, like RT-PCR, 5'-RACE and cDNA library screening.Can clone and check order through pcr amplification from the V gene of the MAb of the cell of expressing muroid MAb.For confirming its reliability, can make clone's V LAnd V HGene is expressed as chimeric Ab in cell culture, like said (Proc.Natl.Acad.Sci., USA, 86:3833 (1989)) such as Orlandi.Based on the V gene order, then can be like (Mol Immunol, 32:1413 (1995)) said design and structure humanization MAb such as Leung.
Can prepare cDNA (Sambrook etc., Molecular Cloning, A laboratory manual, the 2nd edition (1989)) from any known hybridoma cell line or the transfectional cell series that produces muroid MAb through general molecule clone technology.The V of MAb κSequence can use primer VK1BACK and (Bio Techniques, 15:286 (1993)) described expansion primer sets such as VK1FOR (Orlandi etc., 1989) or Leung to increase.V HSequence can use primer to increase with the annealed primer of muroid IgG constant region to (Hybridoma, 13:469 (1994)) such as VH1BACK/VH1FOR (Orlandi etc., 1989) or Leung is described.Can make up humanization V gene through combination long oligonucleotide masterplate is synthetic with pcr amplification as (Mol.Immunol, 32:1413 (1995)) such as Leung is said.
V κBut PCR product sub-clone in the support carrier, like the support carrier VKpBR based on pBR327, it contains Ig promoter, signal peptide sequence and suitable restriction site.V HBut PCR product sub-clone in similar support carrier, like VHpBS based on pBluescript.Contain V κAnd V HSequence can and be connected to respectively the suitable expression vector from VKpBR and VHpBS excision together with the expression cassette of promoter and signal peptide sequence, like pKh and pG1g (Leung etc., Hybridoma, 13:469 (1994)).But in said expression vector cotransfection to the suitable cell and chimeric, the humanization or the people MAb that monitor supernatant produce.Perhaps, can excise V κAnd V HExpression cassette and sub-clone are to single expression vector, like pdHL2, like Gillies etc. said (J Immunol.Methods 125:191 (1989), and at Losman etc., Cancer is shown in the 80:2660 (1997)).
In an alternate embodiment, can be with the expression vector transfection to adapting in the host cell of transfection in serum-free medium, growth and expression in advance.Spendable exemplary cells is to comprise that Sp/EEE, Sp/ESF and Sp/ESF-X cell line are (referring to for example United States Patent(USP) No. 7,531,327; 7,537,930 and 7,608,425; Embodiment part is separately incorporated this paper by reference into).These exemplary cells system is based on saltant Bcl-EEE gene transfection, is exposed to methotrexate with amplification institute's rotaring redyeing gene sequence and adapt to the Sp2/0 myeloma cell line of serum-free cell line for protein expression in advance.
Butt joint locking (DNL)
In certain embodiments, the targeted delivery complex can use the manufacturing of butt joint lock-in techniques (referring to for example United States Patent(USP) No. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400, the embodiment part of each patent is incorporated this paper by reference into).The DNL method is utilized specific protein/protein interaction (Baillie etc., the FEBS Letters.2005 that takes place between the anchoring structure territory (AD) of regulation and control (R) subunit and A-kinases anchorin (AKAP) of cAMP deopendent protein kinase (PKA); 579:3264.Wong and Scott, Nat.Rev.Mol.Cell Biol.2004; 5:959).Maximum second message,second messenger cAMP that pass through are incorporated into the PKA that plays central action in one of signal transduction path that the R subunit triggers and at first isolate (Walsh etc., J.Biol.Chem.1968 from rabbit skeletal muscle in nineteen sixty-eight in research; 243:3763).The structure of holoenzyme is formed (Taylor, J.Biol.Chem.1989 by two catalytic subunits that remain inactive form through the R subunit; 264:8443).Find that the isozyme of PKA has two types R subunit (RI and RII), and all types ofly have α and β hypotype (Scott, a Pharmacol.Ther.1991; 50:123).The R subunit only is separated into to be stablized dimer and shows that the dimerization domain forms (Newlon etc., Nat.Struct.Biol.1999 by preceding 44 aminoterminal residues; 6:222).CAMP combines to cause discharging the active catalytic subunit of wide spectrum activity of serine/threonine kinases with the R subunit, it makes the PKA compartmentation be defined in selected substrate (Scott etc., J.Biol.Chem.1990 through PKA is docked with AKAP; 265; 21561).
(Lohmann etc., Proc.Natl.Acad.Sci USA.1984 since first kind of AKAP MAP-2 characterized in 1984; 81:6723); Identify the 50 kinds of AKAP that surpass again the species in from yeast to people's scope with multiple structure; It is positioned various subcellular fractions site; Comprise plasma membrane, actin cytoskeleton, nucleus, mitochondrion and endoplasmic reticulum (Wong and Scott, Nat.Rev.Mol.Cell Biol.2004; 5:959).The AD that is used for PKA among the AKAP is amphipathic helix (Carr etc., the J.Biol.Chem.1991 with 14-18 residue; 266:14188).The aminoacid sequence of AD is very different between indivedual AKAP, the binding affinity (Alto etc., the Proc.Natl.Acad.Sci.USA.2003 in 2 to 90nM scopes that are wherein reported to the RII dimer; 100:4445).AKAP only combines dimerization R subunit.For people RII α, hydrophobic surface (Colledge and Scott, Trends Cell Biol.1999 that the AD combination is formed by 23 aminoterminal residues; 6:216).Therefore, the dimerization domain of people RII α and AKAP binding structural domain all are positioned at N and hold (Newlon etc., Nat.Struct.Biol.1999 in the same 44 amino acid whose sequences; 6:222; Newlon etc., EMBO are J.2001; 20:1651), it is called as DDD at this paper.
We have developed and a kind of platform technology; It utilizes the AD of DDD and the AKAP of people RII α to be used for any two entities (being called A and B hereinafter) being connected into non-covalent complex as a pair of good connexon module, and said complex can form disulfide bond and further be locked as the stability series stud structure promoting through in the vital position of DDD and AD, introducing cysteine residues.The conventional method of " butt joint locking " method is following.Entity A makes up through the presoma that the DDD sequence is connected to A, produces first component of a hereinafter referred to as.Because the DDD sequence will realize dimeric spontaneous formation, so A will be by a 2Constitute.Entity B makes up through the presoma that the AD sequence is connected to B, produces second component of b hereinafter referred to as.a 2In the dimerization primitive of contained DDD generation is used for combining the butt joint site of the contained AD sequence of b, promote a thus 2Easily associate formation by a with b 2The binary trimerization complex that b constitutes.Utilization makes this binding events irreversible through the subsequent reactions of two entities of disulphide bridges Covalent Immobilization; Principle based on effective local concentration; This reaction takes place extremely effectively; Because initial binding interactions should make the reactive mercapto that is placed on DDD and the AD near (Chmura etc., Proc.Natl.Acad.Sci.USA.2001; 98:8480) thereby locus specificity connects.Use the various combinations of connexon, joint module and presoma, can produce and use the multiple DNL construct of different chemical metering, include but not limited to that dimerization, trimerization, four gather, five gather and six gather the DNL construct (referring to for example U.S.No.7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400.)
Functional group through away from two presomas connects DDD and AD, expects that said locus specificity connects the original activity that also will keep two presomas.The method is modular and can potentially be applied to the covalently bound multiple material of locus specificity in nature, comprises peptide, protein, antibody, antibody fragment and has other effect part of various active.Utilize the fusion rotein method of the effector that makes up coupling AD described in following examples and DDD, can be with almost any protein or peptide are incorporated in the DNL construct.Yet said technology not tool is restricted and can utilize other coupling method.
Become known for preparing the several different methods of fusion rotein, comprise that nucleic acid synthesizes, hybridizes and/or increases to produce the proteic synthetic double-strandednucleic acid of coding Target Fusion.Can said double-strandednucleic acid be inserted through standard molecular biological technique and be used for making Expression of Fusion Protein carrier (referring to for example Sambrook etc., Molecular Cloning, A laboratory manual, the 2nd edition, 1989).In said preferred embodiment, AD and/or DDD part can be connected in the N-end or the C-end of effect protein or peptide.Yet those of skill in the art will recognize, the site that AD or DDD partly are connected in the effect part can change according to the part of its physiologically active of participation in the chemical property of effect part and the effect part.The locus specificity of multiple effect part connects can use technology execution as known in the art, as using bivalence cross-linking reagent and/or other chemical coupling technology.
Immune conjugate
In preferred embodiments, can with as parts such as siRNA carrier part covalently bound to antibody or antibody fragment to form immune conjugate.Can carrier part be connected to and for example pass through reductive SH group and/or carbohydrate side chain.Carrier part can be connected in the hinge region through reductive antibody component through forming disulfide bond.Perhaps, said medicament can use Heterobifunctional cross-linking agent (like 3-(2-pyridine radicals disulfide group) propanoic acid N-succinyl ester (SPDP)) to connect.Yu etc., Int.J.Cancer 56:244 (1994).Said link coupled general technology is known in the art.Referring to for example Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etc., " Modification of Antibodies by Chemical Methods, " MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS, Birch etc. (writing), the 187-230 page or leaf (Wiley-Liss, Inc.1995); Price; " Production and Characterization of Synthetic Peptide-Derived Antibodies; " MONOCLONAL ANTIBODIES:PRODUCTION; ENGINEERING AND CLINICAL APPLICATION, Ritter etc. (writing), 60-84 page or leaf (Cambridge University Press 1995).Perhaps, can be through the carbohydrate part coupling carrier part in the antibody Fc district.
Method through antibody carbohydrate part coupling functional group and antibody is known by those skilled in the art.Referring to for example Shih etc., Int.J.Cancer41:832 (1988); Shih etc., Int.J.Cancer 46:1101 (1990); With Shih etc., United States Patent(USP) No. 5,057,313, the embodiment part of said document is incorporated this paper by reference into.Conventional method relates to makes antibody with carbohydrate oxidation using part and the carrier polymer reaction with at least one unhindered amina functional group.This reaction produces initial schiff bases (Schiff base) (imines) binding, and it can be stablized to form final conjugate through being reduced into secondary amine.
Be not have the Fc district under the situation of antibody fragment at the antibody component of immune conjugate.Yet, might carbohydrate partly be introduced in the variable region of light chain of full length antibody or antibody fragment.Referring to for example Leung etc., J.Immunol.154:5919 (1995); United States Patent(USP) No. 5,443,953 and 6,254,868, the embodiment part of said document is incorporated this paper by reference into.Use through engineered carbohydrate and partly connect therapeutic agent or diagnostic agent.
The connection carrier part relates to the reaction of use click chemistry with the alternative method of targeted molecular.The click chemistry method is contemplated at first through with modular mode small subunit being bonded together and produces the method for complex material fast.(referring to for example Kolb etc., 2004, Angew Chem Int Ed 40:3004-31; Evans, 2007, Aust J Chem 60:384-95.) reaction of various forms of click chemistries is known in the art, like Hu Yisigen (Huisgen) 1,3-dipole cycloaddition copper catalytic reaction (Tornoe etc., 2002, J Organic Chem 67:3057-64), it is commonly referred to " click-reaction ".Other replacement scheme comprises cycloaddition reaction; Carbonylation like Di Ersi-Alder (Diels-Alder), nucleophilic substitution (especially with like small strain rings such as epoxy and azacyclopropane chemical compounds reacting), carbamide compound forms and relates to the reaction of carbon-carbon double bond, like the alkynes in mercaptan-alkyne reaction.
Azide alkynes Hu Yisigen cycloaddition reaction uses the copper catalyst under the Reducing agent existence to come catalysis to be connected in the reaction of the terminal alkynyl of first molecule.In the presence of second molecule that comprises the azide part, azide and activation alkyne reaction form 1, the substituted 1,2,3-triazoles of 4-.The catalytic reaction of copper is at room temperature carried out and is had enough specificitys, thereby need not carry out purification to product usually.(Rostovstev etc., 2002, Angew Chem Int Ed 41:2596; Tornoe etc., 2002, J Org Chem 67:3057.) nitrine and alkynes functional group be inertia basically to the biomolecule in the aqueous medium, carry out thereby allow to be reflected in the complicated solution.The triazole that forms makes the click chemistry product stable at the biosystem camber chemically for stable and do not experience enzymatic lysis.Although copper catalyst is poisonous to living cells, can be at the click chemistry reaction formation immune conjugate of external use based on copper.
Having proposed does not have the covalent modification that the copper click-reaction is used for biomolecule.(referring to for example Agard etc., 2004, J Am Chem Soc 126:15046-47.) reaction of no copper uses ring strain instead of copper catalyst to promote [3+2] azide-alkynes cycloaddition reaction (the same).For example, cyclooctyne is the 8 carbocyclic ring structures that comprise inner acetylene bond.Closed-loop construct is brought out the substantive bond-angle deformation of acetylene, and it very easily forms triazole with the azido reaction.Therefore, the cyclooctyne derivant can be used for not having copper click-reaction (the same).
The no copper click-reaction of another type is by reports (2010, Angew Chem Int Ed 49:3065-68) such as Ning, and it relates to alkynes-nitrone cycloaddition that strain promotes.For solving original cyclooctyne reaction rate problem slowly, the adjacent electron withdraw group (the same) that is connected with triple bond.The instance of the substituted cyclooctyne of said process comprises bifluoride cyclooctyne, the pure and mild azacyclo-octyne of 4-dibenzo cyclooctyne (the same).Alkynes-nitrone cycloaddition that one alternative no copper reaction relates to the strain promotion obtains N-alkylation isoxazoline (the same).Said reaction has express kinetics according to reports and is used for a cooking-pot type three steps scheme for peptide and protein are carried out site-specific sex modification (the same).Nitrone is through suitable aldehyde and the condensation of N-methyl hydroxylamine being prepared and cycloaddition reaction is carried out (the same) in the mixture of acetonitrile and water.Can use these and other known click chemical reaction carrier part to be connected to antibody external.
The method of therapeutic treatment
Various embodiments relate to the method for cancer of treating the experimenter, and it comprises the targeted delivery complex of administering therapeutic effective dose, as with the antibody of coupling of siRNA carrier and load siRNA molecule.
The targeted delivery complex can replenish with the while or use at least a other therapeutic agent in regular turn.The multi-mode therapy can comprise the therapy of utilizing other antibody, as is the anti-CD22 of naked antibody, fusion rotein or immune conjugate form, anti-CD19, anti-CD20, anti-CD21, anti-CD74, anti-CD80, anti-CD23, anti-CD45, anti-CD46, anti-MIF, anti-EGP-1, anti-CEACAM5, anti-CEACAM6, anti-cancer of pancreas mucin, anti-IGF-1R or anti-HLA-DR (comprising constant chain) antibody.Employed various antibody, like anti-CD19, anti-CD20 and anti-CD22, for those skilled in the art institute known.Referring to for example Ghetie etc., Cancer Res.48:2610 (1988); Hekman etc., Cancer Immunol.Immunother.32:364 (1991); Longo, Curr.Opin.Oncol.5:353 (1996); United States Patent(USP) No. 5,798,554; 6,187,287; 6,306,393; 6,676,924; 7,109,304; 7,151,164; 7,230,084; 7,230,085; 7,238,785; 7,238,786; 7,282,567; 7,300,655; 7,312,318; 7,612,180; 7,501,498; Embodiment part is separately incorporated this paper by reference into.
In another form of multi-mode therapy, the experimenter accepts the associating of targeted delivery complex and standard cancer chemotherapy.For example, " CVB " (1.5g/m 2Cyclophosphamide, 200-400mg/m 2Etoposide and 150-200mg/m 2Carmustine) is the scheme that is used to treat the non-Hodgkin lymphomas.Patti etc., Eur.J.Haematol.51:18 (1993).Other suitable chemical treating composition scheme is known by those skilled in the art.Referring to for example Freedman etc., " Non-Hodgkin's Lymphomas, " CANCER MEDICINE, the 2nd volume, the 3rd edition, Holland etc. (writing), 2028-2068 page or leaf (Lea&Febiger 1993).For instance, the first generation chemotherapy scheme that is used to treat intergrade non-Hodgkin lymphomas (NHL) comprises C-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide, amycin, vincristine and prednisone).Being suitable for second filial generation chemotherapy scheme is m-BACOD (methotrexate, bleomycin, AC, vincristine, dexamethasone and formyl tetrahydrofolic acid), is MACOP-B (methotrexate, AC, vincristine, prednisone, bleomycin and formyl tetrahydrofolic acid) and be fit to third generation scheme.Other suitable medicine comprises phenyl butyrate, bendamustine and bryostatin-1.
In a preferred multi-mode therapy, chemotherapeutic agent and cytokine are used with the targeted delivery complex simultaneously jointly.Cytokine, chemotherapeutic agent and targeted delivery complex can any order or are used together.
The targeted delivery complex can be prepared the compositions that pharmaceutically is suitable for preparation according to known method, thus with targeted delivery complex and the vehicle group synthetic mixture that pharmaceutically is fit to.SPBS is an instance of the excipient that pharmaceutically is fit to.Other suitable excipient is known by those skilled in the art.Referring to for example Ansel etc.; PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS; The 5th edition (Lea & Febiger 1990); And Gennaro (writing), REMINGTON'S PHARMACEUTICAL SCIENCES, the 18th edition (Mack Publishing Company 1990) and its revised edition.
Targeted delivery complex of the present invention can be mixed with and be used for through for example injecting or continuous infusion carries out intravenous administration.Preferably, targeted delivery complex infusion continues to be less than about 4 hours time, and more preferably continues to be less than about 3 hours time.For example, preceding 25-50mg can be at 30 minutes, preferred in addition 15 minutes in infusion, and remainder is followed infusion and is continued 2-3 hour.The preparation that is used to inject can unit dosage forms, for example in ampoule that is added with antiseptic or multi-dose container, provides.Compositions can adopt such as forms such as the suspension in oiliness or aqueous vehicles, solution or emulsions, and can contain formula agent, like suspensoid, stabilizing agent and/or dispersant.Perhaps, active component can be before use with being fit to the restorative powder type of mediator (for example aseptic apyrogeneity matter water).
The targeted delivery complex also can be through subcutaneous or even be applied to mammal through other parenteral approach.In addition, can inject through continuous infusion or through single or multiple and use.Preferably, targeted delivery complex infusion continues to be less than about 4 hours time, and more preferably continues to be less than about 3 hours time.
More in general, the dosage that is used for people's the targeted delivery complex of using will change according to following factor, like patient's age, body weight, height, sex, general curative condition and previous medical history taking.Need to the dosage of the targeted delivery complex that the receiver provides as the single intravenous infusion time in about 1mg/kg to 25mg/kg scope, but also can use lower or higher dosage according to environmental requirement.Dosage for 70kg patient 1-20mg/kg for example is 70-1,400mg, or be 41-824mg/m for 1.7-m patient 2Dosage can repeat when needing, and for example continues 4-10 week once in a week, continues for 8 weeks once in a week, or continues for 4 weeks once in a week.When in maintenance therapy, needing, it can also give by lower frequency, as once continuing several months week about, or every month or per season once continue many months,
Perhaps, the targeted delivery complex can be used dose in per 2 or 3 weeks, repeated at least 3 dosage altogether.Perhaps, the targeted delivery complex can be used weekly and continue 4-6 week for twice.Reduce to about 200-300mg/m if make dosage 2(for 1.7-m patient is every dose of 340mg, is 4.9mg/kg for 70kg patient perhaps), it can be used weekly once or even continue for 4 to 10 weeks twice so.Perhaps, the administration time-histories can reduce, and promptly per 2 or 3 weeks once continued 2-3 month.Yet confirmed, even can be that weekly or every 2-3 week once like 20mg/kg through slow intravenous infusion according to repeat administration cyclical administration even higher dosage.The administration time-histories can be randomly repeating at interval At All Other Times, and dosage can time give through various parenteral approach suitably adjusting dosage and time-histories.
In preferred embodiments, target targeted delivery complex is to be used to treat cancer.The instance of cancer includes but not limited to carcinoma, lymphoma, glioblastoma, melanoma, sarcoma and leukemia, myeloma or lymph malignant disease.The more particular instance of said cancer is described below and comprises: squamous cell carcinoma (for example epithelium squamous cell carcinoma); Ewing's sarcoma (Ewing sarcoma); Wilms' tumor (Wilms tumor); Astrocytoma; Pulmonary carcinoma (comprises small cell lung cancer; Nonsmall-cell lung cancer; Adenocarcinoma of lung and lung squamous cell carcinoma); Peritoneal cancer; Hepatocarcinoma; Stomach cancer or gastric cancer (comprising human primary gastrointestinal cancers); Cancer of pancreas; Glioblastoma multiforme; Cervical cancer; Ovarian cancer; Hepatocarcinoma; Bladder cancer; Hepatoma; The hepatocyte carcinoma; Neuroendocrine tumor; Medullary thyroid carcinoma; Differentiated thyroid carcinoma; Breast carcinoma; Ovarian cancer; Colon cancer; Rectal cancer; Carcinoma of endometrium or uterus carcinoma; Salivary-gland carcinoma; Renal cancer or renal carcinoma; Carcinoma of prostate; The vaginal orifice cancer; Anus cancer; Carcinoma of penis and head and neck cancer.Term " cancer " comprises primary malignancy cell or tumor (for example cell does not migrate to the tumor at original malignant disease in the subject or the position beyond the tumor locus) and Secondary cases malignant cell or tumor (for example by shifting the tumor that produces, shifting is the extremely secondary position different with original tumor locus of malignant cell or tumor cell migration).
Cancer or other malignant diseases include, but are not limited to: acute lymphoblastic leukemia children, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, adrenal cortical carcinoma, adults (primary) liver cell cancer, adult (primary) liver cancer, adult acute lymphoblastic leukemia, adult acute myeloid leukemia, Hodgkin's lymphoma, adult, adult lymphoblastic lymphoma, adult non-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS related malignancies, anal cancer, astrocytoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumors, breast cancer, renal pelvis and ureter cancer, central nervous system (Primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, children (primary) hepatocellular carcinoma, children (primary sex) liver cancer, children with acute lymphoblastic leukemia, acute myelogenous leukemia of children, child brain stem glioma, cerebellar astrocytoma children, children's brain astrocytoma, extracranial germ cell tumors of children, child Hodge Guinness disease, Hodgkin's lymphoma, children, children's hypothalamus and visual pathway glioma, children lymphoblastic leukemia, children as medulloblastoma, a children's non-Hodgkin's lymphoma, a children's pineal gland and supratentorial primitive neuroectodermal tumors, children with primary liver cancer, rhabdomyosarcoma children, children soft tissue sarcoma, a children's visual pathway and hypothalamic glioma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, skin T-cell lymphoma, endocrine islet cell cancer, endometrial cancer, ependymoma, epithelial cancer, esophageal cancer, Ewing's sarcoma and related tumors, exocrine pancreatic cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, female breast cancer, Gaucher disease (Gaucher's? Disease), gallbladder cancer, stomach cancer, benign gastrointestinal, gastrointestinal tumors, germ cell tumors, gestational trophoblastic tumor, hair cells leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, high agammaglobulinemia, hypopharyngeal cancer, colon cancer, intraocular melanoma, pancreatic islet cell carcinoma, pancreatic islet cells, Kaposi's sarcoma (Kaposi's? Sarcoma), kidney cancer, laryngeal cancer, lip cancer, liver cancer, lung cancer, lymphoproliferative disorders, macroglobulinemia, male breast cancer, malignant mesothelioma, malignant thymoma, as medulloblastoma, Black melanoma, mesothelioma, occult primary tumor in metastatic squamous neck cancer, metastatic primary squamous cervical carcinoma, metastatic squamous neck cancer, multiple myeloma, multiple myeloma / plasma cell neoplasia , myelodysplastic syndrome, myeloid leukemia, myeloid leukemia, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma and non-melanoma skin cancer , non-small cell lung cancer, primary foci of occult metastatic squamous neck cancer, oropharyngeal cancer, osteosarcoma / malignant fibrosarcoma, osteosarcoma / malignant fibrous histiocytoma, osteosarcoma / malignant fibrous histiocytoma of bone tumors, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, lesions albumin, polycythemia vera, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumors, primary central nervous system lymphoma , primary liver cancer, prostate cancer, rectal cancer, renal cell carcinoma, carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, sarcoidosis, Sezary Syndrome (Sezary? Syndrome), skin carcinoma, small cell lung cancer, small bowel cancer, soft tissue sarcoma, squamous neck cancer, stomach cancer, supratentorial primitive neuroectodermal and pineal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, renal pelvis and ureter transitional cell carcinoma, transitional carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis carcinoma, ureter cancer, uterine cancer, uterine sarcoma, vaginal cancer, depending on the pathway and hypothalamic glioma, vulva cancer, Waldenstrom macroglobulinemia, Vail Williams' tumor, and in addition to the above neoplasia away in any other organ system in hyperproliferative diseases.
This paper describe and the method and composition of advocating can be used for treating pernicious or cancerate before condition of illness and be used for prevention and develop into superfluous natural disposition or malignant state, include but not limited to those diseases mentioned above.Said purposes is applicable to known or suspects the condition of illness that develops into anything superfluous or useless or cancer in advance; Especially under the situation of the non-neoplastic cell growth that takes place to form, (about the summary of said misgrowth condition of illness, see also Robbins and Angell, Basic Pathology by hypertrophy, conversion or the most particularly abnormal development; The 2nd edition; W.B.Saunders Co., Philadelphia, 68-79 page or leaf (1976)).
Abnormal development usually is the omen of cancer, and mainly is found in the epithelium.The ordered form of its right and wrong neoplastic cell growth relates to the forfeiture of individual cells concordance and cellularity orientation.Under the situation that has chronic stimulation or inflammation, abnormal development characteristic ground takes place.Medicable abnormal development disease includes but not limited to: anhidrotic ectodermal dysplasia; The front face dysplasia; Asphyxiating thoracic dysplasia; Atriodigital dysplasia; Broncho-pulmonary dysplasia; Brain development is bad; The cervical atypism hypertrophy; Chondroectodermal dysplasia; Cleidocranial dysplasia; Congenital ectodermal dysplasia; Skull is done dysplasia; Cranio-carpo-tarsal dysplasia; Craniometaphyseal dysplasia; Dentinal dysplasia; Diaphysial dysplasia; Ectodermal dysplasia; The enamel dysplasia; Encephalo-ophthalmic dysplasia; Dysplasia epiphysialis hemimelia; Multiple epiphyseal dysplasia; Chondrodystrophia congenita punctata; Epithelial dysplasia; Face refers to that genital development is unusual; Familial fibrous dysplasia of jaw; Familial white gauffer sexual abnormality; Fibrillar muscle abnormal development; Fibrous dysplasia of bone; Florid osseous dysplasia; Heritability kidney retinal dysplasia; Hidrotic ectodermal dysplasia; Hypohidrotic ectodermal dysplasia; Lymphopenia property thymic hypoplasia; Mammary gland dysplasia; The dysplasia of mandibular bone face; Metaphyseal dysplasia; Cover base of a fruit Nissl abnormal development (Mondini dysplasia); Monostotic fibrous dysplasia; Mucous epithelium abnormal development; Multiple epiphyseal dysplasia; OAVD; Oculodentodigital dysplasia; Oculovertebral dysplasia; The odontogenic dysplasia; Jaw growth is bad now; Periapical cemental dysplasia; Polyostotic fibrous dysplasia; Pseudoachondroplasia property spondylo epiphyseal dysplasia; Retinal dysplasia; In separated-eye dysplasia; The radially dysplasia of the spondylo epiphyseal dysplasia and the ventricles of the brain.
Medicable other superfluous disease before death includes but not limited to optimum paraplasm sexually transmitted disease (STD) disease (for example benign tumor, Fibrocystic disease shape, tissue hypertrophy, polyp intestinal or adenoma and esophagus paraplasm), leukoplakia, keratosis, Bowen's disease (Bowen's disease), farmer's skin (Farmer's Skin), solar cheilitis and solar keratosis.
In preferred embodiments, use method of the present invention to suppress cancer, the particularly growth of the listed cancer of preceding text, development and/or transfer.
Other hyperplasia property disease, disease and/or condition of illness include but not limited to the progress and/or the transfer of malignant disease and associated conditions; Like leukemia (comprising acute leukemia (for example acute lymphoblastic leukemia, acute cellulous leukemia of bone marrow (comprising skeletonization myeloid, promyelocyte property, bone marrow mononuclear cell, monocarpotic cellularity and erythroleukemia)) and chronic leukemia (for example chronic medullary cell property (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (for example lymphogranulomatosis and Fei Huoqijinshi are sick), multiple myeloma, macroglobulinemia Waldenstron, heavy chain disease and entity tumor; Include but not limited to sarcoma and carcinoma, like fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, Wilms' tumor, cervical cancer, carcinoma of testis, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.
Other therapeutic agent
Multiple therapeutic agent can or be used with the targeted delivery complex while in regular turn.For example medicine, toxin, oligonucleotide, immunomodulator, hormone, hormone antagonist, enzyme, enzyme inhibitor, radionuclide, angiogenesis inhibitor etc.Therapeutic agent described herein is those medicaments that also are applicable to aforesaid targeted delivery complex separate administration.Therapeutic agent comprises for example chemotherapeutic agent; Like vinca alkaloids, anthracycline, gemcitabine, taxane, antimetabolite, alkylating agent, antibiotic, SN-38, cox 2 inhibitor, antimitotic agent, anti-angiogenic agent and short apoptosis agent, particularly amycin, methotrexate, taxol, CPT-11, camptothecine, proteasome inhibitor, mTOR inhibitor, hdac inhibitor, tyrosine kinase inhibitor and other medicines.
Other suitable cancer chemotherapeutic medicine comprises chlormethine, alkylsulfonate, nitroso ureas, triazenes, folacin, cox 2 inhibitor, antimetabolite, pyrimidine analogue, purine analogue, platinum coordination complex, mTOR inhibitor, tyrosine kinase inhibitor, proteasome inhibitor, hdac inhibitor, camptothecine, hormone etc.Suitable chemotherapeutant is described in the following document: REMINGTON'S PHARMACEUTICAL SCIENCES; The 19th edition (Mack Publishing Co.1995) and GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS; The 7th edition (MacMillan Publishing Co.1985), and the revised edition of these publications.Other suitable chemotherapeutant (like experimental drug) is that those skilled in the art institute is known.
In a preferred embodiment, can be with camptothecine and related compound (like SN-38) and anticancrin coupling, for example like United States Patent(USP) No. 7,591,994; With disclosed among the USSN 11/388,032 that submitted on March 23rd, 2006, the embodiment part of said each document is incorporated this paper by reference into.
Toxin can have animal, plant or microbe-derived.Toxin (like PE) is also can be with the therapeutic agent part of immune conjugate compound or form the therapeutic agent part of immune conjugate.Other toxin comprises Ricin, abrin, ribonuclease (RNA enzyme), DNA enzyme I, staphyloentero-toxin-A, PAP, antitumor ribonuclease (onconase), gelonin, diphtheria toxin, diphtherotoxin, PE and pseudomonas endotoxin.Referring to for example Pastan etc., Cell 47:641 (1986), Goldenberg, CA--ACancer Journal for Clinicians 44:43 (1994); Sharkey and Goldenberg, CA--A Cancer Journal for Clinicians 56:226 (2006).Other toxin that is fit to use is known and be disclosed in United States Patent(USP) No. 6,077 as those skilled in the art institute, and in 499, the embodiment of said patent partly incorporates this paper by reference into.
As used herein, term " immunomodulator " comprises that (IFN, parathormone, thyroxine, insulin, proinsulin, relaxin, relaxation precipitinogen, follicle stimulating hormone (FSH), thyrotropin (TSH), lutropin (LH), LGF, prostaglandin, fibroblast growth factor, prolactin antagonist, galactagogin, OB albumen, transforming growth factor (TGF), TGF-α, TGF-β, insulin like growth factor (IGF), erythropoietin, thrombopoietin, tumor necrosis factor (TNF), TNF-α, TNF-β, Seedling are reined in pipe inhibiting substances (mullerian-inhibiting substance), mice gonadotropin related peptides, inhibin, activin, VEGF, integration element, interleukin (IL), granulocyte colony stimulating factor (G-CSF), granular leukocyte macrophage group stimulating factor (GM-CSF), interferon-' alpha ', interferon-beta, interferon-, the S1 factor, IL-1, IL-1cc, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21 and IL-25, LIF, kit-part, FLT-3, angiostatin, thrombospondin, Endostatin and LT etc. for cytokine, lymphokine, monokine, stem cell factor, lymphotoxin, Hemopoietic factor, group's stimulating factor (CSF), interferon.
The targeted delivery complex can be used with comprising one or more radioisotopic immune conjugates that are applicable to treatment illing tissue.The therapeutic radiation property nucleic of particularly suitable includes but not limited to 111In, 177Lu, 212Bi, 213Bi, 211At, 62Cu, 67Cu, 90Y, 125I, 131I, 32P, 33P, 47Sc, 111Ag, 67Ga, 142Pr, 153Sm, 161Tb, 166Dy, 166Ho, 186Re, 188Re, 189Re, 212Pb, 223Ra, 225Ac, 59Fe, 75Se, 77AS, 89Sr, 99Mo, 105Rh, 109Pd, 143Pr, 149Pm, 169Er, 194Ir, 198Au, 199Au with 211Pb.Therapeutic radiation property nucleic preferably has 20 to 6, the decay in the 000keV scope can, preferably for the Auger emitter in 60 to 200keV scopes; For beta emitter at 100-2, in the 500keV scope, and for alpha emitter 4; 000-6 is in the 000keV scope.The maximum decay that is suitable for beta-particle emission nucleic can be preferably 20-5,000keV, and 100-4 more preferably, 000keV, and most preferably be 500-2,500keV.The radionuclide that decays in fact with the Auger emitted particle also is preferred.For example Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161, Os-189m and Ir-192.The decay that is suitable for beta-particle emission nucleic can be preferably 1,000keV, more preferably < 100keV, and most preferably be < 70keV.The radionuclide of decay also is preferred in fact along with the alpha-particle generation.Said radionuclide includes but not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255.The decay that is suitable for alpha-particle emission radionuclide can be preferably 2,000-10, and 000keV, more preferably 3,000-8,000keV, and most preferably be 4,000-7,000keV.
Other potential therapeutic radiation property isotope comprises 11C, 13N, 15O, 75Br, 198Au, 224Ac, 126I, 133I, 77Br, 113mIn, 95Ru, 97Ru, 103Ru, 105Ru, 107Hg, 203Hg, 121mTe, 122mTe, 125mTe, 165Tm, 167Tm, 168Tm, 197Pt, 109Pd, 105Rh, 142Pr, 143Pr, 161Tb, 166Ho, 199Au, 57Co, 58Co, 51Cr, 59Fe, 75Se, 201Tl, 225Ac, 76Br, 169Yb etc.
The excipient that pharmaceutically is fit to
The targeted delivery complex of desiring to be delivered to the experimenter can comprise excipient, one or more other compositions or its some combinations that one or more pharmaceutically are fit to.The targeted delivery complex can be prepared the compositions that pharmaceutically is suitable for preparation according to known method, thus with targeted delivery complex and the vehicle group synthetic mixture that pharmaceutically is fit to.SPBS is an instance of the excipient that pharmaceutically is fit to.Other suitable excipient is known by those skilled in the art.Referring to for example Ansel etc.; PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS; The 5th edition (Lea & Febiger 1990); And Gennaro (writing), REMINGTON'S PHARMACEUTICAL SCIENCES, the 18th edition (Mack Publishing Company 1990) and its revised edition.
The targeted delivery complex can be mixed with and be used for through for example injecting or continuous infusion carries out intravenous administration.The preparation that is used to inject can unit dosage forms, for example in ampoule that is added with antiseptic or multi-dose container, provides.Compositions can adopt such as forms such as the suspension in oiliness or aqueous vehicles, solution or emulsions, and can contain formula agent, like suspensoid, stabilizing agent and/or dispersant.Perhaps, active component can be before use with being fit to the restorative powder type of mediator (for example aseptic apyrogeneity matter water).
Test kit
Various embodiments can relate to containing and are suitable for treating or the test kit of the component of the illing tissue of diagnosing patients.Exemplary kit can contain at least a targeted delivery complex as described herein.Be not mixed with through digestive tract and send if contain the compositions that is useful on the component of using, send like administered through oral, can comprise so can be through the device of some other approach delivery of agents box components.A kind of types of devices that is used for sending etc. like parenteral application is a syringe, and it is used for compositions is injected in the subject.Also can use suction apparatus.In certain embodiments, the targeted delivery complex can contain antibody sterile liquid formulations or lyophilized formulations pre-filled syringe or automatically injection pen form provide (Kivitz etc. for example, Clin.Ther.2006,28:1619-29).
Reagent constituents can be packaged in together or be separated in two or more containers.In some embodiments, container can be the bottle that holds the sterile freeze-drying preparation that is suitable for restorative compositions.Test kit also can contain the buffer that one or more are suitable for restoring and/or diluting other reagent.Spendable other container includes but not limited to bag, dish, box, pipe etc.But reagent constituents aseptic packaging and remaining in the container.Another component that can comprise is the operation instructions of test kit.
Embodiment
Following examples explanations embodiment of the present invention the and scope of claims is not constituted restriction.
Embodiment 1. preparation butt joint locking (DNL) constructs
DDD and AD fusion rotein
Can use the DNL technology to prepare to comprise the dimer, trimer, the tetramer, six aggressiveness of almost any antibody, antibody fragment, siRNA carrier or other effect part etc.For some preferred embodiment, the antibody of target targeted delivery complex and carrier part can be fabricated to the fusion rotein that comprises dimerization and docking structure territory (DDD) or anchoring structure territory (AD) sequence.Although in preferred embodiments; DDD and AD part can be bonded into fusion rotein with targeted molecular and siRNA carrier, but those of skill in the art will recognize, has other coupling method; Particularly for nonprotein siRNA carrier, like chemical crosslinking, click chemistry reaction etc.
Said technology not tool is restricted and can employed any protein or peptide be fabricated to AD or DDD fusion rotein for being incorporated in the DNL construct.Utilizing under the situation of chemical crosslinking, AD and DDD conjugate can comprise any molecule that can use any crosslinking technological as known in the art and AD or DDD sequence crosslinked.In some exemplary, can dendritic or other polymeric part (like PEI or Polyethylene Glycol (PEG)) be incorporated in the DNL construct, describe in further detail like hereinafter.
For dissimilar DNL constructs, different AD capable of using or DDD sequence.Exemplary DDD and AD sequence are provided in hereinafter.
DDD1：SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：33)
DDD2：CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：34)
AD1：QIEYLAKQIVDNAIQQA(SEQ?ID?NO：35)
AD2：CGQIEYLAKQIVDNAIQQAGC(SEQ?IDNO：36)
Those of skill in the art will recognize that DDD1 and DDD2 comprise the DDD sequence of the people RII alpha form of PKA.Yet in alternate embodiment, DDD and AD part can be based on the DDD sequence and the corresponding AKAP sequence of the people RI alpha form of PKA, like institute's illustration among hereinafter DDD3, DDD3C and the AD3.
DDD3
SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK(SEQ?ID?NO：37)
DDD3C
MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK(SEQ?ID?NO：38)
AD3
CGFEELAWKIAKMIWSDVFQQGC(SEQ?ID?NO：39)
Expression vector
Used plasmid vector pdHL2 to produce multiple antibody and based on the construct of antibody.Referring to Gillies etc., JImmunol Methods (1989), 125:191-202; Losman etc., Cancer (Phila) (1997), 80:2660-6.Synthesizing of the heavy chain of said bicistronic mRNA mammalian expression vector guiding IgG and light chain.The carrier sequence of many different I gG-pdHL2 constructs is substantially the same, and only difference is present in variable domains (VH and the VL) sequence.Use biology tool known to those skilled in the art, can these IgG expression vectors be changed into Fab-DDD or Fab-AD expression vector.Be to produce the Fab-DDD expression vector, the coded sequence of hinge, CH2 and the CH3 domain of heavy chain is replaced as the sequence of preceding 44 residues (being called DDD1) of Gly-Ser connexon and the people RII α of preceding 4 residues of coding hinge, 14 residues.For producing the Fab-AD expression vector; The sequence of hinge, CH2 and the CH3 domain of IgG is replaced as the Gly-Ser connexon of preceding 4 residues of coding hinge, 15 residues and is called the sequence of synthetic AD (being called AD1) of 17 residues of AKAP-IS, and said synthetic AD is to use bioinformatics and peptide array technique to produce and confirms the dimer with high affinity (0.4nM) combination RII α.Referring to Proc.Natl.Acad.Sci. such as Alto, U.S.A (2003), 100:4445-50.
Design two shuttle vectors to promote of the conversion of IgG-pdHL2 carrier to Fab-DDD1 or Fab-AD1 expression vector, as mentioned below.
Preparation CH1
Use the pdHL2 plasmid vector to pass through pcr amplification CH1 domain as masterplate.Left side PCR primer is made up of the upper reaches (5') end and SacII restriction endonuclease site (it is the 5' of CH1 coded sequence) of CH1 domain.The right side primer by preceding 4 residues (PKSC) of coding hinge then four glycine form latter two codon (GS) formation Bam HI restriction site wherein with serine.The 410bp? PCR amplification product was cloned into the PCR cloning vector (
Figure BDA00001666436900442
Inc.) and in accordance with T7 (5 ') direction of the filter insert the clone.
Synthetic duplex oligonucleotide is there to be the DDD1 aminoacid sequence of 11 connexon peptide residues before encoding, wherein preceding two codons constitute the BamHI restriction site.Termination codon and EagI restriction site are attached to the 3' end.The encoded polypeptide sequence illustrates in hereinafter.
GSGGGGSGGGG SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：40)
Synthesizing on the 3' end has 30 eclipsed two oligonucleotide of base pair, called after RIIA1-44top and RIIA1-44bottom, and with its combination 154 base pairs in center with formation 174bp DDD1 sequence.Make said oligonucleotide annealing and carry out primer extension reaction with the Taq polymerase.Behind the primer extension, through the pcr amplification duplex.Amplified matter is cloned in
Figure BDA00001666436900443
and and screens according to the insert on T7 (5') direction.
Synthetic duplex oligonucleotide is there to be the AD1 aminoacid sequence of 11 connexon peptide residues before encoding, wherein preceding two codons constitute the BamHI restriction site.Termination codon and EagI restriction site are attached to the 3' end.The encoded polypeptide sequence illustrates in hereinafter.
GSGGGGSGGGG SQIEYLAKQIVDNAIQQA(SEQ?ID?NO：41)
Two overlapping oligonucleotide of complementation of the above-mentioned peptide sequence of composite coding, called after AKAP-ISTop and AKAP-IS Bottom, and anneal.Through the pcr amplification duplex.Amplified matter is cloned in
Figure BDA00001666436900451
carrier and and screens according to the insert on T7 (5') direction.
Connect DDD1 and CH1
With BamHI and NotI restriction enzyme sequence encoding DDD1 190bp fragment from
Figure BDA00001666436900452
excised and then connect to the in the same locus to produce a shuttle vector
Figure BDA00001666436900454
Connect AD1 and CH1
With BamHI and NotI containing AD1 sequence 110bp fragment from
Figure BDA00001666436900455
excised and then connect to the
Figure BDA00001666436900456
in the same locus to produce a shuttle vector
Figure BDA00001666436900457
CH1-DDD1 or CH1-AD1 are cloned in the carrier based on pdHL2
According to this modularized design, can CH1-DDD1 or CH1-AD1 be incorporated in any IgG construct in the pdHL2 carrier.Through removing SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and it being replaced into from the CH1-DDD1 of corresponding pGemT shuttle vector excision or the SacII/EagI restriction fragment of CH1-AD1 whole heavy chain constant domain is replaced into one of above-mentioned construct.
Make up h 679-Fd-AD1-pdHL2
H679-Fd-AD1-pdHL2 be used to make AD1 through the flexible Gly/Ser peptide introns coupling that constitutes by 14 amino acid residues in the expression vector of the h679Fab of the carboxyl terminal of the CH1 of Fd domain.Be converted into h679-Fd-AD1-pdHL2 through the SacII/EagI fragment being replaced into from the carrier that the CH1-AD1 fragment of CH1-AD1-SV3 excision will contain the variable domains of h679 based on pdHL2 with SacII and EagI.
Make up C-DDD 1-Fd-hMN-14-pdHL2
C-DDD1-Fd-hMN-14-pdHL2 is used to make comprise the dimeric expression vector of stablizing of two copy fusion PROTEIN C-DDD1-Fab-hMN-14, and wherein DDD1 is connected in the c-terminus that hMN-14Fab goes up CH1 through flexible peptide introns.Through changing into C-DDD1-Fd-hMN-14-pdHL2 to remove the CH1-CH3 domain and to insert plasmid vector hMN-14 (I)-pdHL2 that the CH1-DDD1 fragment of excising from the CH1-DDD1-SV3 shuttle vector with SacII and EagI will be used to make hMN-14IgG with the digestion of SacII and EagI restriction endonuclease.
Utilized the constructed plasmid of making the Fab expression that is used for multiple known antibodies, said antibody such as hLL1, hLL2, hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and a lot of other antibody.In general, the antibody variable region coded sequence is present in the pdHL2 expression vector and transforms expression vector as stated and is used to make AD or DDD fusion rotein.The segmental AD of Fab and the DDD fusion rotein that can comprise any said antibody by the roughly ratio combination of corresponding two the DDD fusion rotein of each AD fusion rotein comprise two Fab fragments of first antibody and the segmental trimerization DNL of a Fab construct of SA with generation.
Make up N-DDD1-Fd-hMN-14-pdHL2
N-DDD1-Fd-hMN-14-pdHL2 is used to make the dimeric expression vector of stablizing that comprises two copy fusion albumen N-DDD1-Fab-hMN-14, and wherein DDD1 is connected in the aminoterminal that hMN-14Fab goes up VH through flexible peptide introns.Expression vector carries out engineered as follows.Through pcr amplification DDD1 domain.
As the result of PCR, respectively NcoI restriction site and the part coded sequence that contains the restrictive connexon of BamHI are attached to 5' and 3' end.170bp pcr amplification thing is cloned in the pGemT carrier and according to the insert on T7 (5') direction clone is screened.With NcoI and SalI restriction endonuclease from pGemT carrier excision 194bp insert and be cloned into through with the SV3 shuttle vector of same enzyme digestion preparation to produce intermediate carrier DDD1-SV3.
Through pcr amplification hMN-14Fd sequence.As the result of PCR, the coded sequence of BamHI restriction site and part connexon is attached to the 5' end of amplified matter.Termination codon and EagI restriction site are attached to the 3' end.The 1043bp amplified matter is cloned among the pGemT.Also then and through DDD1-SV3 carrier be connected from pGemT excision hMN-14-Fd insert with the EagI restriction endonuclease with BamHI to produce construct N-DDD 1-hMN-14Fd-SV3 with same enzyme digestion preparation.
With XhoI and EagI restriction enzyme digestion remove the N-DDD1-hMN-14Fd sequence and with 1.28kb insert fragment be connected through digest the carrier segments that C-hMN-14-pdHL2 prepares with same enzyme.Final expression vector is N-DDD 1-Fd-hMN-14-pDHL2.The demonstration of N-connecting-type Fab fragment forms with the similar DNL complex of C-connecting-type Fab fragment and antigen combines the characteristic (not shown).
CDDD2-Fd-hMN-14-pdHL2
C-DDD2-Fd-hMN-14-pdHL2 is used to make the expression vector of C-DDD2-Fab-hMN-14 of c-terminus that dimerization with DDD2 and the docking structure territory sequence Gly/Ser peptide connexon through 14 amino acid residues is attached to the Fd of hMN-14.Secreted fusion rotein is made up of the hMN-14Fab of two identical copies that the noncovalent interaction through the DDD2 domain keeps together.
Expression vector carries out engineered as follows.Synthetic preparation comprises two overlapping complementary oligonucleotides of coded sequence of the residue 1-13 of part connexon peptide and DDD2.With the annealing of said oligonucleotide and use the T4PNK phosphorylation, overhang with the DNA that digests with restriction endonuclease BamHI and PstI respectively is connected thereby generation is suitable on 5' and 3' end.
The double-stranded DNA and body through digestion with BamHI and PstI prepared shuttle vector
Figure BDA00001666436900471
Connect to produce the shuttle vector
Figure BDA00001666436900472
with SacII and EagI from
Figure BDA00001666436900481
cut 507bp fragment with SacII and EagI digestion by using a prepared IgG expression vector hMN-14 (I)-pdHL2 connection.Final expression construct called after C-DDD2-Fd-hMN-14-pdHL2.Utilized similar techniques to produce the segmental DDD2 fusion rotein of Fab of multiple different humanized antibodies.
h679-Fd-AD2-pdHL2
Design h679-Fab-AD2 as B with as the C-DDD2-Fab-hMN-14 of A pairing.H679-Fd-AD2-pdHL2 is used to make the expression vector of h679-Fab-AD2 that anchoring structure territory sequence with AD2 Gly/Ser peptide connexon through 14 amino acid residues is attached to the carboxyl terminal of CH1 domain.AD2 has a cysteine residues in the anchoring structure territory of AD1 before the sequence and has another cysteine residues thereafter.
Expression vector carries out engineered as follows.Synthetic preparation comprises two overlapping complementary oligonucleotides (AD2Top and AD2Bottom) of the coded sequence of AD2 and part connexon sequence.With the annealing of said oligonucleotide and use the T4PNK phosphorylation, overhang with the DNA that digests with restriction endonuclease BamHI and SpeI respectively is connected thereby generation is suitable on 5' and 3' end.
Connected to the duplex DNA with BamHI and SpeI digested through the preparation of a shuttle vector
Figure BDA00001666436900482
to produce a shuttle vector
Figure BDA00001666436900483
with restriction enzymes SacII and EagI removal from the shuttle vector containing the CH1 and AD2 coding sequences 429 base pair fragment and connect to via digested with the same enzymes prepared h679-pdHL2 vector.Final expression vector is h679-Fd-AD2-pdHL2.
Embodiment 2. produces the TF1DNL construct
The mass preparation that is called the DNL construct of TF1 as follows.At first N-DDD2-Fab-hMN-14 (protein L purification) and h679-Fab-AD2 (IMP-291 purification) are mixed in 1mM EDTA, PBS (pH 7.4) with stoichiometric concentration roughly.Before adding TCEP, SE-HPLC does not show any a 2The sign (not shown) that b forms.Represent a but exist 4(7.97min; 200kDa), a 2(8.91min; 100kDa) and B (10.01min; Peak 50kDa).Add 5mM TCEP and cause a fast 2The b complex forms, during like 8.43min the new peak consistent with 150kDa protein the proof (not shown).In this experiment, obviously there is excessive B, still obviously and not observes corresponding to a because belong to the peak (9.72min) of h679-Fab-AD2 2Or a 4Obvious peak.Reduce after one hour, change the PBS dialysed overnight through several times TCEP is removed.Making gained solution reach 10%DMSO also at room temperature keeps spending the night.
When analyzing, represent a through SE-HPLC 2As if the peak of b is more sharp-pointed, and wherein 0.1min falls in the holdup time slightly becomes the 8.31min (not shown), according to our previous discovery result, this shows that binding affinity increases.Complex is further purified to remove κ chain pollutant through the IMP-291 affinity chromatography.Such as expection, copurification goes out excessive h679-AD2 and is removing (not shown) through preparation type SE-HPLC after a while.
TF1 is high stability complex.When the combining of test TF1 and HSG (I Μ Ρ-239) sensor chip, the reaction that when sample injection end, is observed does not have obvious decline.On the contrary; When test under conditions of similarity contain C-DDD1-Fab-hMN-14 and h679-Fab-AD1 etc. during the solution of molar concentration mixture; During the sample injection, follow obvious decline when reacton increases, show a of initial formation with being right after to observe thereafter 2The b structural instability.In addition, although follow-up injection WI2 makes the reacton of TF1 obtain substantive increasing, C-DDD1/AD1 does not have obvious increase.
The extra increase of the reacton that is combined by WI2 and TF1 on being fixed in sensor chip to cause conforms to two complete functional binding sites that the subunit of each free N-DDD2-Fab-hMN-14 is facilitated.This is combined two Fab (not shown) that segmental ability confirms of WI2 by TF1.Reduce and when accurately being oxidized to the mixture of h679-AD2 and N-DDD1-hMN14 of TF1, have the extra combination (not shown) of few WI2 when analyzing through BIAcore to contain, show a that disulfide bond is stable 2The interaction that b complex (like TF1) possibly only pass through DDD2 and AD2 forms.
The two places improvement of implementation method is with the time and the efficient of minimizing method.At first, use molar excess a little with a 4/ a 2The N-DDD2-Fab-hMN-14 that the structure form of mixtures exists comes the reaction with h679-Fab-AD2 so that do not have free h679-Fab-AD2 residual and not drift bolt in any a of h679-Fab-AD2 4/ a 2Structure and light chain will remove through the IMP-291 affinity chromatography.The second, substitute the means that dialysis or diafiltration remove TCEP after as reduction with hydrophobic interaction chromatograph (HIC), it will not only reduce the method time, remove step but also increase potential virus.Mix N-DDD2-Fab-hMN-14 and 679-Fab-AD2 also at room temperature with 5mM TCEP reduction 1 hour.Making solution reach 0.75M ammonium sulfate also then is loaded on the butyl FF HIC tubing string.Tubing string washs to remove TCEP with 0.75M ammonium sulfate, 5mM EDTA, PBS.Reductive protein with PBS from HIC tubing string eluting and make it reach 10%DMSO.At room temperature after the incubated overnight, through the TF1 (not shown) of IMP-291 affinity chromatography transport disengaging height purification.Do not need like extra purification steps such as gel filtrations.
Embodiment 3. produces the TF2DNL construct
Through making C-DDD2-Fab-hMN-14 and h679-Fab-AD2 reaction obtain trimerization DNL construct, called after TF2.As follows with 90% productive rate produces the test batch of TF2.With the C-DDD2-Fab-hMN-14 (200mg) of protein L purification and h679-Fab-AD2 (60mg) with the 1.4:1 mixed in molar ratio.Total protein concentration is 1.5mg/ml in the PBS that contains 1mM EDTA.Subsequent step comprises TCEP reduction, HIC chromatograph, DMSO oxidation and IMP 291 affinity chromatographies.Before adding TCEP, SE-HPLC does not show any a 2The sign that b forms.Adding 5mM TCEP causes and the consistent a of 157kDa protein that expects to diadactic structure fast 2The b complex forms.Through IMP 291 affinity chromatographies TF2 is purified near the homogenizing (not shown).IMP 291 contains the 679Fab haptenic synthetic peptide of bonded HSG (Rossi etc., 2005, Clin Cancer Res 11:7122s-29s).IMP 291 is the SE-HPLC analytical proof a of bound fraction not 4, a 2With the remove (not shown) of free κ chain from product.
Measure the functional of TF2 through
Figure BDA00001666436900501
algoscopy.TF2, C-DDD1-hMN-14+h679-AD1 (are used as non-covalent a 2The control sample of b complex) or C-DDD2-hMN-14+h679-AD2 (as not reducing a 2Control sample with the b component) is diluted to 1 μ g/ml (gross protein) and flow through and be fixed with the sensor chip of HSG.The reaction of TF2 is about the twice of the reaction of two control samples, shows in the contrast that only the h679-Fab-AD component combines and remaines on the sensor chip.The anti-id AB WI2IgG of follow-up injection hMN-14 proves that only TF2 has the tight associating DDD-Fab-hMN-14 component with h679-Fab-AD, and is indicated like another signal reaction.The extra increase of the reacton that is combined by WI2 and TF2 on being fixed in sensor chip to cause conforms to two complete functional binding sites that the subunit of each free C-DDD2-Fab-hMN-14 is facilitated.This is combined two Fab (not shown) that segmental ability confirms of WI2 by TF2.
Embodiment 4. makes the TF10 bi-specific antibody
Use similar scheme to produce and comprise two copy C-DDD2-Fab-hPAM4 and a trimerization TF10DNL construct that copies C-AD2-Fab-679.Cancer targeting antibodies component among the TF10 is to derive from hPAM4, and it is the anti-cancer of pancreas mucin of the humanization MAb that studied in great detail as radioactivity MAb (for example Gold etc., Clin.Cancer Res.13:7380-7387,2007).The hapten component is to derive from h679, and it is the anti-histidyl--succinyl group of humanization-glycine (HSG) MAb.Use as stated about making (anti-CEA) 2The disclosed method of * anti-HSGbsAb TF2 is made TF10 bispecific ([hPAM4] 2* h679) antibody.TF10 has two humanization PAM4Fab and a humanization 679Fab.
In the myeloma cell of stable transfection, express two fusion rotein (hPAM4-DDD and h679-AD2) independently.Combination tissue's culture supernatant obtains the hPAM4-DDD of twice molar excess.Reactant mixture was at room temperature hatched 24 hours under the week reduction condition of using the 1mM reduced glutathion.After the reduction, the DNL reaction is accomplished through using 2mM oxidized form of glutathione weak oxide.Use IMP 291-affigel resin isolation TF10 through affinity chromatography, said resin combines h679Fab with high specific.
Those of skill in the art will recognize, can use the disclosed DNL technology of preceding text to make and comprise the complex that antibody, immune conjugate, siRNA carrier part maybe can be connected in other effect any combination partly of AD or DDD part.
Embodiment 5. makes Fab and the IgG fusion rotein that AD is connected with DDD by multiple antibody
Use the technology of describing in the previous embodiment, IgG shown in the structure table 2 and Fab fusion rotein and incorporate in the DNL construct.Fusion rotein keeps the antigen combination characteristic of parental antibody and the antigen-binding activity of DNL construct demonstration is incorporated into antibody or antibody fragment.
Table 2. comprises the fusion rotein of IgG or Fab
Fusion rotein Binding specificity
C-AD1-Fab-h679 HSG
C-AD2-Fab-h679 HSG
C-(AD) 2-Fab-h679 HSG
C-AD2-Fab-h734 Indium-DTPA
C-AD2-Fab-hA20 CD20
C-AD2-Fab-hA20L CD20
C-AD2-Fab-hL243 HLA-DR
C-AD2-Fab-hLL2 CD22
N-AD2-Fab-hLL2 CD22
C-AD2-IgG-hMN-14 CEACAM5
C-AD2-IgG-hR1 IGF-1R
C-AD2-IgG-hRS7 EGP-1
C-AD2-IgG-hPAM4 MUC
C-AD2-IgG-hLL?1 CD74
C-DDD1-Fab-hMN-14 CEACAM5
C-DDD2-Fab-hMN-14 CEACAM5
C-DDD2-Fab-h679 HSG
C-DDD2-Fab-hA19 CD19
C-DDD2-Fab-hA20 CD20
C-DDD2-Fab-hAFP AFP
C-DDD2-Fab-hL243 HLA-DR
C-DDD2-Fab-hLL1 CD74
C-DDD2-Fab-hLL2 CD22
C-DDD2-Fab-hMN-3 ?CEACAM6
C-DDD2-Fab-hMN-15 ?CEACAM6
C-DDD2-Fab-hPAM4 MUC
C-DDD2-Fab-hR1 IGF-1R
C-DDD2-Fab-hRS7 EGP-1
N-DDD2-Fab-hMN-14 ?CEACAM5
The sequence variants of embodiment 6.DNL
In some preferred embodiment, AD and the DDD sequence incorporated in the DNL construct comprise the aminoacid sequence like the disclosed AD1 of preceding text, AD2, AD3, DDD1, DDD2, DDD3 or DDD3C.Yet, in alternate embodiment, the sequence variants of AD capable of using and/or DDD part in the structure of DNL complex.For example, only there are four kinds of variants in people PKADDD sequence, corresponding to the DDD part of PKA RI α, RII α, RI β and RII β.RII α DDD sequence is the basis of disclosed DDD1 of preceding text and DDD2.Four kinds of people PKA DDD sequences illustrate in hereinafter.The DDD sequence is represented the residue 1-44 of RII α, the residue 1-44 of RII β, the residue 12-61 of RI α and the residue 13-66 of RI β.(it should be noted that the sequence of DDD1 partly compares slight change with people PKA RII α DDD.)
PKA?RIα
SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK(SEQ?ID?NO：42)
PKA?RIβ
SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENRQILA?(SEQ?ID?NO：43)
PKA?RIIα
SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ(SEQ?ID?NO：44)
PKA?RIIβ
SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER(SEQ?ID?NO：45)
The structure-function relationship of AD and DDD domain is studied.(referring to for example Burns-Hamuro etc., 2005, Protein Sci 14:2982-92; Carr etc., 2001, J Biol Chem 276:17332-38; Alto etc., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etc., 2006, Biochem J 396:297-306; Stokka etc., 2006, Biochem J 400:493-99; Gold etc., 2006, Mol Cell 24:383-95; Kinderman etc., 2006, Mol Cell 24:397-408, the whole text of said each document is incorporated this paper by reference into.)
For example; Kinderman etc. (2006) have checked the crystal structure of AD-DDD binding interactions and have reached a conclusion; People DDD sequence contain a plurality of dimer form or AKAP combine in important conservative amino acid residues, underline demonstration among the SEQ ID NO:33 hereinafter.(referring to Kinderman etc., Fig. 1 of 2006 incorporates this paper by reference into.) those of skill in the art will recognize, when the sequence variants of design DDD sequence, hope is avoided changing any residue that underlines, and combine not too crucial residue can carry out the conserved amino acid replacement with AKAP for dimerization.
SH IQ IPPG LTE LLQG YT VE VLRQQPPD LVE FA VE YFTR LREARA(SEQ?ID?NO：33)
Alto etc. (2003) have carried out bioinformatic analysis with design RII selectivity AD sequence to the proteic AD sequence of various AKAP, are called AKAP-IS (SEQ ID NO:35), are 0.4nM to the binding constant of DDD.The AKAP-IS sequential design becomes AKAP and the bonded peptide antagonists of PKA.Replace to tend to make in the AKAP-IS sequence and in SEQ ID NO:35, underline demonstration with the residue that combines to weaken of DDD.Those of skill in the art will recognize, when the sequence variants of design AD sequence, hope avoided changing any residue that underlines, and replace and combine not too crucial residue can carry out conserved amino acid for DDD.
The AKAP-IS sequence
QIEYL AKQ IVDN AIQQA(SEQ?ID?NO：35)
Gold (2006) utilizes crystallography and peptide screening to develop SuperAKAP-IS sequence (SEQ ID NO:46), shows for the selectivity of the RII hypotype of PKA 5 magnitudes up to the RI hypotype.Underlined residue is indicated the DDD position that combines enhanced aminoacid replacement partly feasible with respect to AKAP-IS and RII α.In this sequence, N-end Q residue is numbered residue numbering 4 and C-end A residue is a residue numbering 20.Can replace with influence is residue 8,11,15,16,18,19 and 20 (Gold etc., 2006) for the residue of the affinity of RII α.Be expected in some alternate embodiment, available SuperAKAP-IS sequence replaces AKAP-IS AD partial sequence and prepares the DNL construct.Other alternative sequence that can be used to replace AKAP-IS AD sequence is in shown in the SEQ ID NO:47-49.Replacement with respect to the AKAP-IS sequence underlines demonstration.Expection is as the AD2 sequence shown in the SEQ ID NO:46, and AD partly also can comprise extra N-end residue cysteine and glycine and C-end residue glycine and cysteine.
SuperAKAP-IS
QIEY VAKQIVD YAI HQA(SEQID?NO：46)
Substituting AKAP sequence
QIEY KAKQIVD HAI HQA(SEQ?ID?NO：47)
QIEY HAKQIVD HAI HQA(SEQ?ID?N?O：48)
QIEY VAKQIVD HAI HQA(SEQ?ID?NO：49)
Fig. 2 of Gold etc. is open from proteic other DDD binding sequence of multiple AKAP, and is as follows.
RII specificity AKAPs
AKAP-KL
PLEYQAGLLVQNAIQQAI(SEQ?ID?NO：5O)
AKAP79
LLIETASSLVKNAIQLSI(SEQ?ID?NO：51)
AKAP-Lbc
LIEEAASRIVDAVIEQVK(SEQ?ID?NO：52)
RI specificity AKAPs
AKAPce
ALYQFADRFSELVISEAL(SEQ?ID?NO：53)
RIAD
LEQVANQLADQIIKEAT(SEQ?ID?NO：54)
PV38
FEELAWKIAKMIWSDVF(SEQ?ID?NO：55)
Dual specificity AKAPs
AKAP7
ELVRLSKRLVENAVLKAV(SEQ?ID?NO：56)
MAP2D
TAEEVSARIVQVVTAEAV(SEQ?ID?NO：57)
DAKAP1
QIKQAAFQLISQVILEAT(SEQ?ID?NO：58)
DAKAP2
LAWKIAKMIVSDVMQQ(SEQ?ID?NO：59)
Stokka etc. (2006) have also developed the bonded competitor of AKAP and PKA, shown in SEQ ID NO:60-62.Peptide antagonists called after Ht31 (SEQ ID NO:60), RIAD (SEQ ID NO:61) and PV-38 (SEQ ID NO:62).The Ht-31 peptide shows higher affinity for the RII hypotype of PKA, and RIAD and PV-38 show higher affinity for RI.
Ht31
DLIEEAASRIVDAVIEQVKAAGAY(SEQID?NO：6O)
RIAD
LEQYANQLADQIIKEATE(SEQ?ID?NO：61)
PV-38
FEELAWKIAKMIWSDVFQQC(SEQ?IDNO：62)
Hundsrucker etc. (2006) have developed bonded other peptide competitor of AKAP and PKA, are low to moderate 0.4nM with the binding constant of the DDD of the RII form of PKA.The sequence of various AKAP antagonistic peptides is provided in to be replicated in the hereinafter table 3 in the table 1 of Hundsrucker etc.AKAPIS represents a kind of synthetic RII subunit binding peptide.All other peptides all derive from the RII binding structural domain of specified AKAP.
Table 3.AKAP peptide sequence
Peptide sequence
AKAPIS QIEYLAKQIVDNAIQQA(SEQ?ID?NO：35)
AKAPIS-P QIEYLAKQIPDNAIQQA(SEQ?ID?NO：63)
Ht31 KGADLIEEAASRIVDAVIEQVKAAG(SEQ?ID?NO：64)
Ht31-P KGADLIEEAASRIPDAPIEQVKAAG(SEQ?ID?NO：65)
AKAP7δ-wt-pep PEDAELVRLSKRLVENAVLKAVQQY(SEQ?ID?NO：66)
AKAP7δ-L304T-pep PEDAELVRTSKRLVENAVLKAVQQY(SEQID?NO：67)
AKAP7δ-L308D-pep PEDAELVRLSKRDVENAVLKAVQQY(SEQ?ID?NO：68)
AKAP7δ-P-pep PEDAELVRLSKRLPENAVLKAVQQY(SEQ?ID?NO：69)
AKAP7δ-PP-pep PEDAELVRLSKRLPENAPLKAVQQY(SEQ?ID?NO：70)
AKAP7δ-L314E-pep PEDAELVRLSKRLVENAVEKAVQQY(SEQ?ID?NO：71)
AKAP1-pep EEGLDRNEEIKRAAFQIISQVISEA(SEQ?ID?NO：72)
AKAP2-pep LVDDPLEYQAGLLVQNAIQQAIAEQ(SEQ?ID?NO：73)
AKAP5-pep QYETLLIETASSLVKNAIQLSIEQL(SEQ?ID?NO：74)
AKAP9-pep LEKQYQEQLEEEVAKVIVSMSIAFA(SEQ?ID?NO：75)
AKAP10-pep NTDEAQEELAWKIAKMIVSDIMQQA(SEQ?ID?NO：76)
AKAP11-pep VNLDKKAVLAEKIVAEAIEKAEREL(SEQ?ID?NO：77)
AKAP12-pep NGILELETKSSKLVQNIIQTAVDQF(SEQ?ID?NO：78)
AKAP14-pep TQDKNYEDELTQVALALVEDVINYA(SEQ?ID?NO：79)
Rab32-pep ETSAKDNINIEEAARFLVEKILVNH(SEQ?ID?NO：80)
At the conservative residue of the proteic AD domain of different AKAP camber hereinafter through underlining demonstration with reference to AKAP IS sequence (SEQ ID NO:35).(2003) such as residue and Alto are observed identical to the person, wherein are added with C-end alanine residue.(, incorporate this paper by reference into referring to the Fig. 4 of (2006) such as Hundsrucker.) peptide antagonists of high affinity of sequence have to(for) RII DDD sequence is the sequence of AKAP-IS, AKAP7 δ-wt-pep, AKAP7 δ-L304T-pep and AKAP7 δ-L308D-pep.
AKAP-IS
QIEYL AKQ IVDN AIQQ A(SEQ?ID?NO：35)
Carr etc. (2001) have checked the degree that combines sequence homology between the DDD sequence from the different AKAP of people and non-human protein's matter, and identify as if topnotch is guarded in different DDD parts in the DDD sequence residue.It underlines expression through the people PKA RII α DDD sequence with reference to SEQ ID NO:33 hereinafter.Conservative especially residue is further represented with italic.Said residue is with overlapping but inequality for residue important with the AKAP protein binding by proposition such as Kinderman (2006).Those of skill in the art will recognize; When the sequence variants of design DDD; Most preferably avoid changing the most conservative residue (italic), and preferably also avoid changing conserved residues (underlining), and both do not underlined neither italic residue can consider to carry out conserved amino acid and replace.
S HIQ IPP GLT ELLQGYTV EVLRQ QPP DLVEFAVE YFTR LR EA RA(SEQ?ID?NO：33)
Those of skill in the art will recognize that these in the antibody moiety of DNL construct or the connexon part and other aminoacid replacement can be used to strengthen the treatment and/or the pharmacokinetic property of gained DNL construct.
Embodiment 7. is used for antibody-dendritic DNL complex of siRNA
Cation property copolymer like the dendritic based on polylysine, PEI or polyamide-amide (PAMAM), forms complex with nucleic acid.Yet still there is challenge in it as the potential application that is used for the non-virus carrier of delivery of therapeutic gene or siRNA.The method of a kind of selectivity of improveing the dendritic nanoparticle and usefulness can be through reaching with the antibody coupling of internalization when combining target cell.
We are synthetic and characterized a kind of novel immune conjugate; Called after E1-G5/2, it is to be connected in the stabilisation dimer that derives from the Fab of the humanized antibody hRS7 of quick internalization when being bonded to the Trop-2 antigen that is expressed on the various entity tumors through the preparation of DNL method to comprise half the 5th generation (G5) PAMAM dendritic (G5/2) locus specificity.
Method
Through two self assembly modules A D2-G5/2 of combination and hRS7-Fab-DDD2 under the weak oxide reducing condition, then on protein L tubing string, carrying out purification prepares E1-G5/2.Be preparation AD2-G5/2, we with dimaleoyl imino to the AD2 peptide carry out derivatization with single thiol reactant through producing with cystamine core reduction G5PAMAM, and use anti-phase HPLC separates AD2-G5/2.We are fabricated to fusion rotein with hRS7-Fab-DDD2 in the myeloma cell, described in preceding text embodiment.
Analyze molecular size, purity and the composition of E1-G5/2 through size exclusion HPLC, SDS-PAGE and protein immunoblotting.Through with to the combining of the anti-id AB of hRS7, gel blocking is measured and the DNA enzyme protection is measured the biological function of assessing E1-G5/2.
The result
Confirm that through size exclusion HPLC E1-G5/2 is had the main peak (> 90% of some secondary peaks by side joint) form.Three key components (Fd-DDD2, light chain and AD2-G5/2) of E1-G5/2 detect through reproducibility SDS-PAGE and are confirmed through protein immunoblotting.The anti-idiotype binding analysis discloses antibody-dendritic conjugate that E1-G5/2 contains the different sizes of a group, all can discern anti-id AB, shows that therefore the G5 dendritic of buying has the structural change property of size.Gel blocking measure to show that E1-G5/2 can farthest concentrate DNA with the charge ratio of 6:1 (+/-), and wherein gained dendritic complex (dendriplex) protect compound DNA to exempt from DNA enzyme I to degrade fully.
Conclusion
Can use the DNL technology to set up the nanoparticle based on dendritic of available antibodies targeting.Said medicament has the character of improvement as the carrier of the medicine, plasmid or the siRNA that are used for using in vitro and in vivo.
The maleimide AD2 conjugate of embodiment 8.DNL dendritic
IMP?498(SEQ?ID?NO:35)
Figure BDA00001666436900601
Utilize Protein Technologies PS3 peptide synthesizer through the Fmoc method Xi Baier amide resin (Sieber Amide resin) go up synthetic peptide IMP 498 until and comprise peg moiety (0.1mmol scale).β-dimaleoyl imino propanoic acid NHS the ester and the resiniferous DMF that contain diisopropylethylamine through mixing continue 4 hours manually interpolation maleimides.With 15mLTFA, 0.5mL H 2O, 0.5mL tri isopropyl silane and 0.5mL thio phenyl methyl ether make peptide from the resin cracking.Use H through anti-phase HPLC 2O/CH 3CN TFA buffer purified peptide obtains about 90mg purified product after the lyophilizing.
Synthetic reduced form G5 dendritic (G5/2)
Use 0.1426TCEP.HCl 1:1MeOH/H 2O (about 4mL) reductase 12 .03g7.03 * 10 -6Mol G-5 dendritic (among the MeOH 10%, Dendritic Nanotechnologies) and stirred overnight at room temperature.On the C-18 tubing string, use 0.1%TFA H through anti-phase HPLC 2O/CH 3The CN buffer solution elution is carried out purification to reactant mixture, obtains 0.0633g expectation product after the lyophilizing.
Synthetic G5/2 dendritic-AD2 conjugate
With 0.0469g (3.35 * 10 -6Mol) G5/2 dendritic and 0.0124g (4.4 * 10 -6Mol) IMP 498 mixes and is dissolved in 1:1MeOH/1M NaHCO 3In and at room temperature mixed 19 hours, then with 0.0751g dithiothreitol, DTT and 0.0441g TCEP HCl processing.Solution at room temperature mixes and spends the night and on C4 anti-phase HPLC tubing string, use 0.1%TFA H 2O/CH 3CN buffer purification judges that according to gel electrophoresis and protein immunoblotting acquisition 0.0033g contains the material of link coupled AD2 and dendritic.
Embodiment 9. uses the antibody target that connects protamine to send siRNA
General introduction
Shown that in preclinical study RNA disturbs the expression of (RNAi) downward modulation like range proteins such as HER2, VEGF, Raf-1, bcl-2, EGFR and numerous other protein.Although RNAi has the potential of reticent specific gene, whole treatment potential of RNAi still for want of are used for being delivered to effective delivery system of target cell in vivo and have to be achieved.
For solving this crucial needs, we developed people's protamine drift bolt with a plurality of copies in the novel DNL construct of cancer target internalization hRS7 (anti-Trop-2) antibody for targeted delivery siRNA in vivo.Manufacturing comprises the DDD2-L-thP1 module of truncate people protamine (thP1, the residue 8 to 29 of people's protamine 1), and wherein the sequence of DDD2 and thP1 is blended in the terminal and C-terminal (Fig. 1) of N-of humanized antibody light chain respectively.The sequence of truncate hP1 (thP1) illustrates hereinafter.Described in above embodiment, DDD2-L-thP1 and antibody hRS7-IgG-AD2 react under the weak oxide reducing condition and cause forming the E1-L-thP1 complex (Fig. 1) that the thP1 that comprises four copies is connected in the c-terminus of hRS7 heavy chain.
tHP1
RSQSRSRYYRQRQRSRRRRRRS(SEQ?ID?NO：81)
Purity and molecule integrity (not shown) through E1-L-thP1 behind size exclusion HPLC and the SDS-PAGE mensuration a-protein purification.In addition, measure the ability (Fig. 2) that proof E1-L-thP1 combines DNA or siRNA through gel shift.E1-L-thP1 effectively combine short double chain oligonucleotide (Fig. 2) with protect bonded DNA exempt from and be added in the sample or be present in the nuclease digestion (not shown) in the serum.
Use the siRNA of coupling FITC and people Calu-3 lung cancer cell line to confirm that the E1-L-thP1 construct makes the ability (Fig. 3) in siRNA internalization to the cancerous cell of expressing Trop-2 through fluorescence microscopy.
Method
Adopt DNL technique for generating E1-L-thP1 (Fig. 1).In the myeloma cell, express the hRS7IgG-AD module (Figure 1A) that described in above embodiment, makes up and use the a-protein affinity chromatography from the culture supernatants purification.DDD2-L-thP1 module (Figure 1B) is expressed as fusion rotein and carries out purification through protein L affinity chromatography in the myeloma cell.Because the CH3-AD2-IgG module has two AD2 peptides and can combine the DDD2 dimer separately, wherein each DDD2 monomer is connected in the protamine part, so gained E1-L-thP1 conjugate comprises four protamine groups (Fig. 1 C).E1-L-thP1 by the component module to form near quantitative yield and to be purified near the homogenizing (not shown) with a-protein.
Use protein L affinity chromatography purification DDD2-L-thP1 and measure (data not shown goes out) through the SDS-PAGE under size exclusion HPLC analysis and reduction and the non-reduced condition.Observe the main peak (not shown) at 9.6min.SDS-PAGE be presented in the reproducibility gel main band 30 and 40kDa between, and main band is about 60kDa (dimeric forms of indication DDD2-L-thP1) (not shown) in the irreducibility gel.The result of protein immunoblotting confirms when surveying with anti-DDD antibody, to exist monomer DDD2-L-tP1 and dimer DDD2-L-tP1 (not shown).
Be preparation E1-L-thP1, with hRS7-IgG-AD2 and DDD2-L-thP1 with about equally amount combination and interpolation reduced glutathion (ultimate density 1mM).At room temperature after the incubated overnight, add oxidized form of glutathione (ultimate density 2mM) and also continued again to hatch 24 hours.Through a-protein tubing string chromatograph from reactant mixture purification E1-L-thP1 and with 0.1M sodium citrate buffer solution (pH 3.5) eluting.The neutralized reaction product peak concentrates, and with the PBS dialysis, filters and under 2 to 8 ℃, is stored among the PBS that contains 5% glycerol.Through the composition (not shown) of reproducibility SDS-PAGE confirmation E1-L-thP1, show to have all three kinds of components (heavy chain of attached AD2, DDD2-L-htP1 and light chain).
Measure the ability of assessment DDD2-L-thP1 (not shown) and E1-L-thP1 (Fig. 2) combination DNA through gel shift.DDD2-L-thP1 is 6 or blocks the migration (not shown) of 500ng linear forms 3-kb DNA in 1% agarose when higher in mol ratio.It is 4 or the migration (Fig. 2, swimming lane d-g) of blocking the linear 200-bpDNA duplex of 250ng in 2% agarose when higher in mol ratio that Fig. 2 illustrates E1-L-thP1, and (Fig. 2, swimming lane a) and when only hRS7-IgG-AD2 being arranged, do not observe said effect.E1-L-thP1 protection combine DNA to exempt from endogenous dna enzyme and serum nuclease degradation ability also obtain proving (not shown).
The ability (Fig. 3) of inspection E1-L-thP1 siRNA internalization that promotion combines in expressing the ME-180 cervical cell system of Trop-2.The internalization of E1-L-thP1 complex uses goat anti people's antibody of coupling FITC to monitor (Fig. 3 a, left side down).Separate cell does not show fluorescence (Fig. 3, upper left).Add the internalization (Fig. 3, upper right) that causes the siRNA minimum level through the siRNA of FITC labelling separately.The internalization of E1-L-thP1 was being observed (Fig. 3 a, left side down) in 60 minutes under 37 ℃ separately.E1-L-thP1 can effectively promote the internalization (Fig. 3, bottom right) of siRNA of bonded coupling FITC.E1-L-thP1 (10 μ g) is mixed with FITC-siRNA (300nM) and lets it form the E1-L-thP1-siRNA complex, then be added in the Calu-3 cell of expressing Trop-2.After hatching 4 hours under 37 ℃, through the siRNA internalization (Fig. 3, bottom right) of fluorescence microscopy inspection cell.
Check E1-L-thP1 is through the ability (Fig. 4) of siRNA internalization cell death inducing.(Valencia CA) mixes for AllStars Cell Death siRNA, Qiagen with not commensurability siRNA with E 1-L-thP1 (10 μ g).The E1-L-thP1-siRNA complex is added in the ME-180 cell.After hatching 72 hours, cell is with trypsin treatment and carry out annexin V dyeing (annexin V staining) with assessment apoptosis (Fig. 4).Cell Death siRNA or independent E1-L-thP1 pair cell apoptosis do not have influence (Fig. 4) separately.The E1-L-thP1-siRNA that adds incremental change causes the apoptosis dose dependent to increase (Fig. 4).The result shows the apoptosis that E1-L-thP1 can effectively be delivered to the siRNA molecule in the cell and induce target cell.
Conclusion
The DNL technology provides the modular method that effectively thereby a plurality of protamine molecule drift bolts is produced novel molecule E1-L-thP1 in anti-Trop-2hRS7 antibody.
SDS-PAGE shows homogeneity and the purity of E1-L-thP1.DNA enzyme protection and gel shift are measured the dna binding activity that shows E1-L-thP1.E1-L-thP1 also can effectively make siRNA molecule internalization to the cell of expressing Trop-2, like ME-180 and Calu-3 with the same internalization of parent hRS7 antibody in cell.
Those of skill in the art will recognize that the DNL technology is not limited to any antibodies specific or siRNA material.But, can use with method and composition identical shown in this paper to prepare the targeted delivery complex that comprises any antibody, any siRNA carrier and any siRNA material.In the targeted delivery complex, use bivalence IgG will produce the circulating half-life of prolongation and higher combination affinity, thereby making to absorb increase and improved efficacy to target cell.The dead the method for siRNA mediated cell is particularly suitable for the disease invalid like current therapy such as cancer of pancreas.Known cancer of pancreas is expressed CEACAM6 and CD74, and the two is all relevant with the aggressivity of cancer of pancreas.The expression of having reported through RNAi inhibition CD74 or CEACAM6 can stop the tumor growth in culture and the experimental animal models.
Embodiment 10. uses the siRNA to CD74 and CEACAM6 to make cancer of pancreas generation apoptosis
To CD74 (sc-35023, Santa Cruz Biotechnology, Santa Cruz, CA) and the siRNA of CEACAM6 [sense strand 5'-CCGGACAGUUCCAUGUAUAdTdT-3' (SEQ ID NO:82)] obtain from commercial source.To there be justice and antisense siRNA to be dissolved in the 30mM HEPES buffer, and be heated to 90 ℃ and kept 1 minute and under 37 ℃, hatched 60 minutes with formation duplex siRNA to the ultimate density of 20 μ Μ.Duplex siRNA is hatched with the E1-L-thP1 mixing and with BxPC-3 (CEACAM6-siRNA) and Capan2 (CD74-siRNA) cell.After 24 hours, the changes of contents of the mRNA through real-time quantitative PCR assay determination respective egg white matter.Measure CD74 and the proteic level of CEACAM6 through protein immunoblotting analysis and immunohistochemistry.Contrast comprises non-specific siRNA and non-targeting DNL complex 20-L-thP1, and it contains Humanized anti-CD 20 antibody (hA20).
Use product clone algoscopy that CD74 and CEACAM6 expression decreased are measured the influence of pancreatic cancer cell growth.Be coated with shop about 1 * 10 3Individual BxPC-3 cell also uses the E1-L-thP1 that carries CEACAM6-siRNA to handle.Changed culture medium in every 3-4 days and after 14 days, group is fixed with 4% metaformaldehyde solution, dye and count with 0.5% trypan blue (trypan blue).The E1-L-thP1 that CD74-siRNA is carried in use carries out similar experiment to the Capan2 cell.Mensuration is carried the E1-L-thP1 of CEACAM6-siRNA and CD74-siRNA to suppressing the influence of BxPC-3 and Capan2 cell growth.Measuring on cell proliferation through MTS detects.
In female athymism nu/nu mice (5 ages in week, body weight 18-20g), set up two xenograft models.Subcutaneous model has BxPC-3 (ATCC No.CRL-1687) and Capan2 (ATCC No.HTB-80) implants the relative flank of each animal, in case tumor reaches 50mm 3, i.e. initiated process.Positive bit model only has the BxPC-3 cell and after implantation, began in 2 weeks to be handled.
For subcutaneous model, effect and the effect of individually sending CEACAM6-siRNA, CD74-siRNA with E1-L-thP1 or contrasting siRNA that assessment E1-L-thP1 sends the mixture of CEACAM6-siRNA and CD74-siRNA compare.Other contrast is for saline and use the alternative E1-L-thP1 of 20-L-thP1 to send specific siRNA and contrast siRNA.Dosage, time-histories and use to by siRNA 150 μ g/kg, continue 6 weeks and for twice weekly through tail vein injection (table 4).Cell increases in tissue culture, utilizes trypsin/EDTA to collect, and with matrigel again suspendible (1: 1) in 300 μ L, to send 5 * 10 6Individual cell.
Monitor the toxicity sign of animal every day and weigh weekly twice.Measure tumor size weekly and calculate gross tumor volume.
Set up positive bit model as follows.Briefly, anesthesia nude mouse and manufacturing left side abdomen otch.Take out spleen and the pancreas that adheres to tweezers.Then with 50 μ L BxPC-3 cell suspensions (2 * 10 6Individual cell) is injected in the pancreas.Put back in the abdominal cavity spleen and pancreas and close incisions.Begin therapy after implanting for two weeks.Mice uses administration time-histories identical with subcutaneous model and approach to handle with the CEACAM6-siRNA or the contrast siRNA general that are incorporated into E1-L-thP 1 or 20-L-thP 1.Detect animal every day and weigh weekly.
Table 4. has the subcutaneous model of dual tumor
Result of study shows; CEACAM6 and CD74siRNA all through E1-L-thP1DNL construct internalization to pancreatic cancer cell and induce the apoptosis of cancer of pancreas, and the contrast DNL construct with non-targeting anti-CD 20 antibodies fails to induce siRNA to absorb and cancer cell death.
***
Those skilled in the art shows and is prone to know, can carry out various modifications and change to product of the present invention, compositions, method and process.Therefore, be intended to the present invention and contain said modification and change, as long as it belongs in the scope of enclose claims and its equipollent.

Claims (39)

1. targeted delivery complex, it comprises:
A) be connected in one or more from the proteic anchoring structure of AKAP territory (AD) part or with one or more dimerizations and docking structure territory (DDD) antibody or its Fabs partly from PKA (PKA); With
B) one or more siRNA carrier parts, each siRNA carrier are connected in from the DDD part of PKA or from the proteic AD part of AKAP;
Wherein the said DDD of two copies partly forms dimer and combines said AD part to form said targeted delivery complex; And wherein when said antibody or fragment are connected in one or more AD part; Said siRNA carrier is connected in the DDD part; Or when said antibody or fragment were connected in one or more DDD part, said siRNA carrier was connected in the AD part.
2. targeted delivery complex according to claim 1, it further comprises one or more siRNA.
3. targeted delivery complex according to claim 1, it further comprises one or more antisense oligonucleotides.
4. targeted delivery complex according to claim 1, it further comprises one or more DNA genes.
5. targeted delivery complex according to claim 2, wherein said targeted delivery complex comprises at least two different siRNA.
6. targeted delivery complex according to claim 1, wherein said siRNA carrier part is selected from the group of being made up of following: dendritic, protamine, histone, the reducible polycation that contains histidine, cationic comb copolymer, chitosan-thiamine pyrophosphate salt, PEI and polylysine.
7. targeted delivery complex according to claim 6, wherein said dendritic comprise the polymer that is selected from by the following group of forming: PAMAM, polylysine, polypropylene imines, PEI, Polyethylene Glycol and carbon silane.
8. targeted delivery complex according to claim 1, wherein said siRNA carrier part comprises the aminoacid sequence of SEQ ID NO:81.
9. targeted delivery complex according to claim 1, wherein said AD partly has the aminoacid sequence that is selected from by the following group of forming: SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79 and SEQ ID NO:80.
10. targeted delivery complex according to claim 1, wherein said DDD partly has the aminoacid sequence that is selected from by the following group of forming: SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45.
11. targeted delivery complex according to claim 2, wherein said siRNA has the nucleotide sequence that is selected from by the following group of forming: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:82.
12. a method of sending siRNA, antisense oligonucleotide or DNA gene, it comprises:
A) obtain targeted delivery complex according to claim 1;
B) at least one siRNA, antisense oligonucleotide or DNA gene are connected in said complex; With
C) said targeted delivery complex is applied to the experimenter.
13. method according to claim 12, wherein said siRNA or antisense oligonucleotide suppress the expression of disease related gene.
14. method according to claim 12, wherein said siRNA or antisense oligonucleotide suppress the expression of cancer related gene.
15. method according to claim 12, wherein said targeted delivery complex comprises at least two different siRNA.
16. method according to claim 12, wherein said siRNA carrier part is selected from the group of being made up of following: dendritic, protamine, histone, the reducible polycation that contains histidine, cationic comb copolymer, chitosan-thiamine pyrophosphate salt, PEI and polylysine.
17. method according to claim 16, wherein said dendritic comprise the polymer that is selected from by the following group of forming: PAMAM, polylysine, polypropylene imines, PEI, Polyethylene Glycol and carbon silane.
18. method according to claim 12, wherein said siRNA carrier part comprises the aminoacid sequence of SEQ ID NO:81.
19. method according to claim 12, wherein said AD partly has the aminoacid sequence that is selected from by the following group of forming: SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79 and SEQ ID NO:80.
20. method according to claim 12, wherein said DDD partly has the aminoacid sequence that is selected from by the following group of forming: SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45.
21. method according to claim 12, wherein said siRNA has the nucleotide sequence that is selected from by the following group of forming: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:82.
22. a method of treating disease, it comprises:
A) obtain targeted delivery complex according to claim 1;
B) at least one siRNA, antisense oligonucleotide or DNA gene are connected in said complex; With
C) said targeted delivery complex is applied to the experimenter.
23. method according to claim 22, wherein said targeted delivery complex comprises at least two different siRNA.
24. method according to claim 22, wherein said siRNA carrier part is selected from the group of being made up of following: dendritic, protamine, histone, the reducible polycation that contains histidine, cationic comb copolymer, chitosan-thiamine pyrophosphate salt, PEI and polylysine.
25. method according to claim 24, wherein said dendritic comprise the polymer that is selected from by the following group of forming: PAMAM, polylysine, polypropylene imines, PEI, Polyethylene Glycol and carbon silane.
26. method according to claim 22, wherein said siRNA carrier part comprises the aminoacid sequence of SEQ ID NO:81.
27. method according to claim 22, wherein said AD partly has the aminoacid sequence that is selected from by the following group of forming: SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79 and SEQ ID NO:80.
28. method according to claim 22, wherein said DDD partly has the aminoacid sequence that is selected from by the following group of forming: SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45.
29. method according to claim 22, wherein said siRNA has the nucleotide sequence that is selected from by the following group of forming: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:82.
30. method according to claim 22, wherein said disease is selected from the group of being made up of following: cancer, autoimmune disease, immune dysfunction, graft versus host disease, organ transplant rejection, inflammation, infectious disease, heart disease and sacred disease.
31. method according to claim 30, wherein said cancer is selected from the group of being made up of following: non-Hodgkin lymphomas (non-Hodgkin ' s lymphoma), B cell acute lymphatic leukemia, B cell chronic lymphatic leukemia, Burkitt lymphoma (Burkitt lymphoma), hodgkin's lymphomas, hairy cell leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, t cell lymphoma, T HTLV, multiple myeloma, glioma, macroglobulinemia Waldenstron (Waldenstrom ' s macroglobulinemia), carcinoma, melanoma, sarcoma, glioma, skin carcinoma, oral cancer, colon cancer, gastric cancer, colon cancer, pulmonary carcinoma, breast carcinoma, ovarian cancer, carcinoma of prostate, uterus carcinoma, carcinoma of endometrium, cervical cancer, bladder cancer, cancer of pancreas, osteocarcinoma, hepatocarcinoma, renal carcinoma and carcinoma of testis.
32. method according to claim 30, wherein said autoimmune disease is selected from the group of being made up of following: acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea (Sydenham ' s chorea), myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyadenous property syndrome, epidermolysis class sky bag skin ulcer, diabetes, Heng Nuoke-Si Qilaien purpura (Henoch-Schonlein purpura), post-streptococcal infection nephritis, erythema nodosum, high iS-One arteritis (Takayasu ' sarteritis), Addison's disease (Addison ' s disease), rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture syndrome (Goodpasture ' s syndrome), thromboangitis obliterans, Xiu Gelun syndrome (Sjogren ' s syndrome), primary biliary cirrhosis, struma lymphomatosa (Hashimoto ' s thyroiditis), thyroxine disease, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, Wei Genashi granulomatosis (Wegener ' s granulomatosis), membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis and fibrosing alveolitis.
33. being cancer and said antibodies, method according to claim 22, wherein said disease be selected from antigen: carbonic anhydrase IX, α-fetoprotein, α-actinine-4, A3, to the specific antigen of A33 antibody tool, ART-4, B7, Ba 733, BAGE, BrE3 antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CXCR4, colon-specific antigen p (CSAp), CEA (CEACAM5), CEACAM6, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3, folacin receptor, G250 antigen, GAGE, gp100, GROB, HLA-DR, HM1.24, human chorionic gonadotropin (HCG), HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, insulin-like growth factor-i (IGF-1), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR/FUT, MIF (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, mucin, placental growth factor, p53, PAP, PSA, PRAME, PSMA, P1GF, IGF, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-α, Tn antigen, thomson-Friedrich antigen (Thomson-Friedenreich antigens), neoplasm necrosis antigen, VEGFR, ED-B fibronectin, WT-1,17-1A antigen, complement factor C3, C3a, C3b, C5a, C5, angiogenesis sign, bcl-2, bcl-6, Kras, cMET and oncogene product by the following group of forming.
34. being cancer and said antibody, method according to claim 22, wherein said disease be selected from the group of forming by following: hR1 (anti-IGF-1R), hPAM4 (anti-stick albumen), hA20 (anti-CD20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEACAM5), hMN-15 (anti-CEACAM6), hRS7 (anti-Trop-2), hMN-3 (anti-CEACAM6), Ab124 (anti-CXCR4) and AM125 (anti-CXCR4).
35. method according to claim 22, it further comprises to said experimenter uses at least a therapeutic agent.
36. method according to claim 35, wherein said therapeutic agent be before the said targeted delivery complex, with said targeted delivery complex simultaneously or after said targeted delivery complex, use.
37. method according to claim 35, wherein said therapeutic agent is selected from the group of being made up of following: radionuclide, chemotherapeutic agent, toxin, immunomodulator, hormone, hormone antagonist, enzyme, photosensitive therapeutic agent, anti-angiogenic agent and short apoptosis agent.
38. a pharmaceutical composition, it comprises targeted delivery complex according to claim 1.
39. a test kit, it comprises targeted delivery complex according to claim 1.
CN2010800526905A 2009-12-09 2010-12-09 Dock-and-lock (DNL) Complexes For Delivery of Interference RNA Pending CN102665758A (en)

Applications Claiming Priority (22)

Application Number Priority Date Filing Date Title
US26787709P 2009-12-09 2009-12-09
US61/267,877 2009-12-09
US12/644,146 US7981398B2 (en) 2005-04-06 2009-12-22 PEGylation by the dock and lock (DNL) technique
US12/644,146 2009-12-22
US30268210P 2010-02-09 2010-02-09
US61/302,682 2010-02-09
US12/731,781 2010-03-25
US12/731,781 US8003111B2 (en) 2005-04-06 2010-03-25 Dimeric alpha interferon pegylated site-specifically shows enhanced and prolonged efficacy in vivo
US12/752,649 2010-04-01
US12/752,649 US8034352B2 (en) 2005-04-06 2010-04-01 Tetrameric cytokines with improved biological activity
US12/754,740 US8562988B2 (en) 2005-10-19 2010-04-06 Strategies for improved cancer vaccines
US12/754,740 2010-04-06
US12/869,823 2010-08-27
US12/869,823 US20110020273A1 (en) 2005-04-06 2010-08-27 Bispecific Immunocytokine Dock-and-Lock (DNL) Complexes and Therapeutic Use Thereof
US12/871,345 US8551480B2 (en) 2004-02-13 2010-08-30 Compositions and methods of use of immunotoxins comprising ranpirnase (Rap) show potent cytotoxic activity
US12/871,345 2010-08-30
US12/915,515 US20110064754A1 (en) 2005-03-03 2010-10-29 Immunoconjugates Comprising Poxvirus-Derived Peptides and Antibodies Against Antigen-Presenting Cells for Subunit-Based Poxvirus Vaccines
US12/915,515 2010-10-29
US41459210P 2010-11-17 2010-11-17
US61/414,592 2010-11-17
US12/949,536 2010-11-18
PCT/US2010/059660 WO2011072114A1 (en) 2009-12-09 2010-12-09 Dock-and-lock (dnl) complexes for delivery of interference rna

Publications (1)

Publication Number Publication Date
CN102665758A true CN102665758A (en) 2012-09-12

Family

ID=44145910

Family Applications (1)

Application Number Title Priority Date Filing Date
CN2010800526905A Pending CN102665758A (en) 2009-12-09 2010-12-09 Dock-and-lock (DNL) Complexes For Delivery of Interference RNA

Country Status (5)

Country Link
EP (1) EP2509629A4 (en)
CN (1) CN102665758A (en)
AU (1) AU2010328136B2 (en)
CA (1) CA2781717C (en)
WO (1) WO2011072114A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102949729A (en) * 2012-11-20 2013-03-06 中国科学院宁波材料技术与工程研究所 Active targeting type small interfering ribonucleic acid (siRNA) delivery carrier and preparation method thereof
CN105168151A (en) * 2015-08-07 2015-12-23 哈尔滨工业大学 Method for preparing oxidation reduction sensitive nanometer targeting carriers
CN107073135A (en) * 2014-09-08 2017-08-18 麦考拉·阿恩特 For the construct being delivered to molecule in the cytoplasm of cell
CN107614685A (en) * 2015-04-17 2018-01-19 肯塔基大学研究基金会 RNA nano particles and its application method
CN112725340A (en) * 2021-01-22 2021-04-30 佛山科学技术学院 Small molecule interference ribonucleic acid and its recombinant adenovirus and application
WO2022206781A1 (en) * 2021-03-30 2022-10-06 南京大学 Rna delivery system for treating colitis

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102472590B1 (en) 2014-05-14 2022-11-30 타르그이뮨 테라퓨틱스 아게 Improved polyethyleneimine polyethyleneglycol vectors

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007127219A2 (en) * 2006-04-25 2007-11-08 Immune Disease Institute, Inc. Targeted delivery to leukocytes using protein carriers
WO2009126558A1 (en) * 2008-04-10 2009-10-15 Ibc Pharmaceuticals, Inc. Modular method to prepare tetrameric cytokines with improved pharmacokinetics by the dock-and-lock (dnl) technology

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7666400B2 (en) * 2005-04-06 2010-02-23 Ibc Pharmaceuticals, Inc. PEGylation by the dock and lock (DNL) technique
US7527787B2 (en) * 2005-10-19 2009-05-05 Ibc Pharmaceuticals, Inc. Multivalent immunoglobulin-based bioactive assemblies
US6958214B2 (en) * 2000-07-10 2005-10-25 Sequenom, Inc. Polymorphic kinase anchor proteins and nucleic acids encoding the same
EP1501863A4 (en) * 2002-05-03 2007-01-24 Sequenom Inc Kinase anchor protein muteins, peptides thereof, and related methods
US7906118B2 (en) * 2005-04-06 2011-03-15 Ibc Pharmaceuticals, Inc. Modular method to prepare tetrameric cytokines with improved pharmacokinetics by the dock-and-lock (DNL) technology
CA2497254A1 (en) * 2002-09-06 2004-03-18 Forschungsverbund Berlin E. V. Akap18 delta, a novel splicing variant of a protein kinase a anchor protein and the use of the same
WO2005076998A2 (en) * 2004-02-05 2005-08-25 Intradigm Corporation Rnai therapeutics for treatment of eye neovascularization diseases
AU2006232310B9 (en) * 2005-04-06 2011-07-21 Ibc Pharmaceuticals, Inc. Improved stably tethered structures of defined compositions with multiple functions or binding specificities
JP5231231B2 (en) * 2005-10-19 2013-07-10 アイビーシー・ファーマシューティカルズ・インコーポレーテッド Methods and compositions and uses for generating bioactive assemblies of increased complexity
KR101410521B1 (en) 2005-12-16 2014-06-20 아이비씨 파마슈티컬스, 인코퍼레이티드 Multivalent immunoglobulin-based bioacitive assemblies
EP2016173A4 (en) * 2006-05-15 2010-06-09 Immunomedics Inc Methods and compositions for treatment of human immunodeficiency virus infection with conjugated antibodies or antibody fragments

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007127219A2 (en) * 2006-04-25 2007-11-08 Immune Disease Institute, Inc. Targeted delivery to leukocytes using protein carriers
WO2009126558A1 (en) * 2008-04-10 2009-10-15 Ibc Pharmaceuticals, Inc. Modular method to prepare tetrameric cytokines with improved pharmacokinetics by the dock-and-lock (dnl) technology

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102949729A (en) * 2012-11-20 2013-03-06 中国科学院宁波材料技术与工程研究所 Active targeting type small interfering ribonucleic acid (siRNA) delivery carrier and preparation method thereof
CN102949729B (en) * 2012-11-20 2014-06-18 中国科学院宁波材料技术与工程研究所 Active targeting type small interfering ribonucleic acid (siRNA) delivery carrier and preparation method thereof
CN107073135A (en) * 2014-09-08 2017-08-18 麦考拉·阿恩特 For the construct being delivered to molecule in the cytoplasm of cell
CN107073135B (en) * 2014-09-08 2023-06-06 鲁塔纳有限公司 Constructs for delivery of molecules into the cytoplasm of cells
CN107614685A (en) * 2015-04-17 2018-01-19 肯塔基大学研究基金会 RNA nano particles and its application method
CN107614685B (en) * 2015-04-17 2021-10-19 肯塔基大学研究基金会 RNA nanoparticles and methods of use thereof
CN105168151A (en) * 2015-08-07 2015-12-23 哈尔滨工业大学 Method for preparing oxidation reduction sensitive nanometer targeting carriers
CN105168151B (en) * 2015-08-07 2017-10-24 哈尔滨工业大学 A kind of preparation method of isotope of redox-sensitive type nano target carrier
CN112725340A (en) * 2021-01-22 2021-04-30 佛山科学技术学院 Small molecule interference ribonucleic acid and its recombinant adenovirus and application
WO2022206781A1 (en) * 2021-03-30 2022-10-06 南京大学 Rna delivery system for treating colitis

Also Published As

Publication number Publication date
AU2010328136A1 (en) 2012-05-17
EP2509629A1 (en) 2012-10-17
CA2781717A1 (en) 2011-06-16
CA2781717C (en) 2017-09-12
WO2011072114A1 (en) 2011-06-16
AU2010328136B2 (en) 2016-02-25
EP2509629A4 (en) 2013-06-26

Similar Documents

Publication Publication Date Title
US9492561B2 (en) Dock-and-Lock (DNL) Complexes for delivery of interference RNA
US9737617B2 (en) Multiple signaling pathways induced by hexavalent, monospecific and bispecific antibodies for enhanced toxicity to B-cell lymphomas and other diseases
JP5870404B2 (en) Dock lock (DNL) vaccine for cancer treatment
AU2011203890B2 (en) Enhanced cytotoxicity of anti-CD74 and anti-HLA-DR antibodies with interferon-gamma
JP5740772B2 (en) Bispecific immune cytokine dock-and-lock (DNL) complex and therapeutic use thereof
US20170088619A1 (en) Antibody-Based Depletion of Antigen-Presenting Cells and Dendritic Cells
US20120276100A1 (en) Compositions and Methods of Use of Immunotoxins Comprising Ranpirnase (Rap) Show Potent Cytotoxic Activity
US20110274704A1 (en) Compositions and Methods of Use of Immunotoxins Comprising Ranpirnase (Rap) Show Potent Cytotoxic Activity
CN104363919A (en) Multimeric complexes with improved in vivo stability, pharmacokinetics and efficacy
JP6114936B2 (en) Compositions and methods of use of immunotoxins containing lampyrnase (RAP) exhibiting potent cytotoxic activity
CN102665758A (en) Dock-and-lock (DNL) Complexes For Delivery of Interference RNA
CN102665757A (en) Delivery system for cytotoxic drugs by bispecific antibody pretargeting
US20130078263A1 (en) Anti-HLA-DR Antibodies Suppress Allogeneic and Xenogeneic Immune Responses to Organ Transplants
US20160375108A1 (en) Compositions and Methods of Use of Immunotoxins Comprising Ranpirnase (Rap) Show Potent Cytotoxic Activity
AU2013203542B8 (en) Enhanced cytotoxicity of anti-CD74 and anti-HLA-DR antibodies with interferon-gamma
AU2013203542B2 (en) Enhanced cytotoxicity of anti-CD74 and anti-HLA-DR antibodies with interferon-gamma

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C12 Rejection of a patent application after its publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20120912